Methods and apparatuses for label-free particle analysis

ABSTRACT

An apparatus to provide a label-free or native particle analysis comprises a light generating system producing first light pulses at a first wavelength and second light pulses at a second wavelength; and a flow cell coupled to the light generating system to convey particles for analysis. The light generating system is configured to chirp at least one of the first light pulses and the second light pulses to analyze the particles.

This application claims priority to U.S. Nonprovisional patentapplication Ser. No. 14/461,293, entitled “METHODS AND APPARATUSES FORLABEL-FREE PARTICLE ANALYSIS,” filed Aug. 5, 2014 and issued on Oct. 3,2017 as U.S. Pat. No. 9,778,193, which claims the benefit of U.S.Provisional Patent Application No. 61/869,035, entitled “METHODS ANDAPPARATUSES FOR LABEL-FREE PARTICLE ANALYSIS,” filed on Aug. 22, 2013,both of which are incorporated herein by reference in their entirety.

FIELD

Embodiments as described herein relate to analysis of particles,specifically to rapid, label-free analysis of particles.

BACKGROUND

Generally, the rapid, accurate, sensitive, and specific analysis ofmicroscopic elements such as cells, microbes, and particulates(hereafter, “particles”) is of great importance in cell biologyresearch, pharmaceutical research and development, microbiology,hematological diagnostics, water and milk quality testing, paint andemulsion production, and other applications. Historically, inspectionand characterization of particles were carried out on opticalmicroscopes, which are still in widespread use in medical, research, andindustrial laboratories worldwide. However, even as microscopes havebecome increasingly sophisticated, with functions such as, e.g.,fluorescence detection, confocal geometry, and superresolution, theycontinue to be severely limited in analysis speed and sample throughput.Collection of image information at high enough resolution to allowcharacterization and identification of human cells is certainlypossible, but at a steep price in terms of the total number ofindividual particles that can be so analyzed in any reasonable amount oftime.

For the last several decades, an alternative modality of analysis,characterization, and counting of particles has emerged and takenroot-flow cytometry. This technology contrasts with microscopy in acouple of key ways: it works by flowing a liquid sample across a singlefixed point of interrogation, rather than interrogating (whether byscanning or imaging) over a fixed sample; and it generally analyzesinformation averaged over each entire particle, rather than resolvingdetails at, e.g., a subcellular or even subnuclear level. The result isa trade-off between spatial resolution and speed: microscopy excels atthe former, flow cytometry at the latter. Accordingly, the twotechniques have in recent years co-evolved by commanding complementaryapplications, often performed in the same laboratory and even side byside: For example, routine hematology diagnostics is near-universallycarried out on highly efficient automated analyzers built on flowcytometric principles, but time-consuming microscope-based review ofblood slides is just as universally performed to clarify byhigh-resolution visual inspection those instances where the flowcytometer results are inconclusive.

Generally, contemporary flow cytometric analysis relies on severalestablished interrogation techniques to extract identifying informationfrom each passing particle: dc electrical impedance, ac electricalconductivity, optical extinction, light scattering, and fluorescence. Dcimpedance is typically very limited in the ability to distinguish cellssimilar in volume but different in composition; it is thereforegenerally used only for first-line hematological screening, or as add-onto optical methods on advanced analyzers. Ac electrical conductivity andoptical extinction can each add a dimension of differentiation to helpdistinguish similar cells, but not in isolation. Typically, theworkhorses of flow cytometry are (elastic) light scattering andfluorescence.

Generally, scattering depends on refractive index variations eitherbetween the particle and its liquid surroundings, or within the particleitself; it gives relatively coarse information on presence, size, andrough morphology of the particle itself and (in the case of a cell) anynucleus and/or cytoplasmic bodies present. Scattering works on intrinsicproperties of particles and does not generally require the use ofexpensive reagents. However, it does not effectively differentiatebetween similarly sized and structured, but functionally distinct,particles (e.g., a lymphocyte and a nucleated erythrocyte; or twomicrobeads surface-functionalized with different analyte-specificantibodies).

Fluorescence is by far the optical interrogation method most widely usedin flow cytometry. A fluorophore can be intrinsic (such as tryptophan);externally introduced as a standalone agent (such as propidium iodide);or externally introduced as a conjugate to a particular antibody (suchas, e.g., phycoerythrin conjugated to CD4). There are furthervariations, such as tandem dyes and quantum dots, which while useful,may not be directly relevant here. Autofluorescence from compoundsnaturally or biologically stimulated within a cell are of interest incertain research areas; however, the broadest application offluorescence in flow cytometry is based on the introduction of externalfluorophores. Non-conjugated fluorophores are typically used to bindselectively to major cell constituents such as DNA or RNA, and returninformation on, e.g., the presence or absence of a nucleus, or on thematuration stage of certain blood cells; but they are not particularlyspecific. They are therefore used in combination with either scattering(in the context, e.g., of hematological diagnosis) or panels offluorescent antibody conjugates.

Antibody-conjugated fluorophores are commonly called “tags” or “labels”in that they perform the function of selectively identifying a particlebased on the presence of matching antigens on the particle surface; alsoused are fluorescent labels that bind to and identify intracellularelements, such as, e.g., actin or cytokeratin. As an example of antibodytagging in peripheral blood samples, leukocytes can be selectivelyidentified out of the much vaster population of erythrocytes, in companyof which they are found, by incubating the sample with a fluorophoreconjugated to the CD45 antibody (which selectively binds to the matchingCD45 antigen on the leukocyte surface). Flow cytometric analysis of theincubated sample results in leukocytes, but not erythrocytes, generatingfluorescence upon passing through the interrogation region. Tagging byantibody conjugation and fluorescence analysis by flow cytometry hasenabled great strides in the field of immunology, where cell type,function, and even stage of development are associated with theexpression of distinct sets of surface antigens.

The development and manufacture of fluorescent antibodyconjugates—particularly monoclonal antibodies (MAbs)—is, however,cumbersome and expensive. While some recent advances have been made inthe generation of synthetic antibodies, most antibodies are producedwith the use of animal-derived cells in a lengthy process. And whileseveral long-established fluorescent dyes are relatively inexpensive,the drive in flow cytometry toward greater and greatermultiplexing—i.e., the concurrent use of multiple tags, and thereforemultiple fluorophores, in the same assay—has pushed the field into agreat proliferation of custom, relatively expensive, fluorophorecompounds. In addition, the process of conjugation itself is anadditional factor in the final cost of the antibody conjugate.

Typical research assays performed on flow cytometers include ones fortransfection, cell signaling, cell lineage, and stem cells; many ofthese assays, especially in immunology, require the concurrent use ofcocktails of large numbers of fluorescent MAb conjugates, resulting inrelatively high costs even for experiments with few repeats. Clinicalpractice is, by contrast, generally focused on the use of relatively fewlabels—for example, in management of AIDS patients, where only two orthree separate MAbs are required for each test—but the relativefrequency of such tests in a typical laboratory is much greater. Ineither research or clinical flow cytometry applications, therefore,expensive reagents used in selective labeling of cells or microparticlesare a definite concern. Annual reagent expenditures in both research andclinical laboratories—expenditures dominated by fluorescent MAbconjugate labels—can be comparable to, and sometimes exceed, the capitalcost of a flow cytometer itself, which typically has a life span of fiveyears or more.

Additionally, there is the issue of fragility. MAbs and MAbs-basedlabels typically have to be kept under strict refrigeration protocols,and thawed carefully just prior to use. This means operationalcomplexity in terms of both proper storage at or near the point of use(hospital or institution) and proper handling during the entire chain ofcustody from manufacturer to end user. In developed countries thiscomplexity is manageable but drives up the cost assays using MAbreagents; in developing countries it often means that those assayscannot be performed. In resource-poor countries, power shortages,unreliable distribution networks, and limited means of refrigeration,not to mention more general financial constraints, make use ofMAb-conjugated fluorophore labels always challenging and oftenimpossible.

Also, whether dealing with MAb tags or with non-conjugated fluorophores,every assay requiring labels involves at least one, and frequently more,incubation steps. Incubation conditions like temperature and time varyconsiderably, but it is common for a single surface-antigen MAbincubation step to take 15 minutes. This delay in the workflow issignificant for a laboratory performing routine tests, and—together withthe high cost of MAbs—has limited widespread adoption of MAb assays forclinical use to those that currently cannot be reliably performed anyother way, like the CD4-positive T-lymphocyte test for AIDS patients.

Moreover, the tolerance toward difficult and time-consuming samplepreparation steps may be relatively high in the context of a clinicallaboratory, and very high in a research laboratory, but it is generallyextremely low in industrial, or industrial-scale, process monitoringsteps, where throughput-limiting (and costly) bottlenecks can mean thedifference between economically viable and nonviable production. Such,for example, is the case of milk quality testing, currently performedmainly in central reference laboratories equipped with high-throughputflow cytometric instruments. Testing for water quality has similarconstraints. In both cases both a high throughput and a low cost perassay are operational imperatives.

In the field of microscopy, certain advances in the use of spectroscopyhave made it possible to analyze specimens without the need forexpensive reagents like MAb-conjugated fluorophore labels. Spectroscopygenerally provides detailed information about the intrinsic chemicalcomposition of a sample by interrogating the sample with opticalradiation. Absorption spectroscopy in its various forms can be verysensitive, but it is not well suited to water-rich samples and aqueoussolutions and suspensions. In such cases, inelastic scatteringspectroscopy techniques are more effective. These techniques differ fromconventional (elastic) light scattering as commonly used in flowcytometry in that they rely on inelastic light scattering—i.e., based onthe transfer of energy from the interrogating photons to the materialunder analysis or vice versa. Raman scattering, resonant Ramanscattering, and Coherent Anti-Stokes Raman Scattering (CARS) areexamples of such techniques that have been successfully applied to thestudy of fixed specimens (as on microscope slides) or, more recently, invivo with small animals. In CARS, pulses at two different opticalfrequencies (ν_(pump) and ν_(Stokes)) are combined at the sample; thenonlinear optical interaction that results from the high peak powers(due to the short pulse duration) is responsible for the CARS signal ata third optical frequency (ν_(anti-Stokes)=2 ν_(pump)−ν_(Stokes)). Inparticular, variations on the CARS technique, such as forward CARS(F-CARS); epi-CARS; polarized CARS (P-CARS); multiplex CARS (M-CARS);broadband CARS (B-CARS) and closely related approaches, such asStimulated Raman Scattering (SRS), have all been demonstrated inbiomicroscopy applications. Each of these techniques is an improvementover traditional microscopy in that it provides detailed informationabout the chemical make-up of the sample, greatly adding specificity tothe analysis.

However, despite advances that enable real-time video-rate analysis of aspecimen, all of these techniques are far too slow, by several orders ofmagnitude, to compete with flow cytometry in the sheer number of cellsor other particles that can be analyzed in a given unit of time.Accordingly, they have overwhelmingly been developed for investigationof stationary samples, and are therefore unsuitable for the kind ofensemble-wide cellular assays provided by flow cytometry.

There have been attempts at combining the superior chemical specificityof spectroscopy with the sample interrogation framework of flowcytometry. For example, experimental results in recent yearsdemonstrated the application of particular versions of CARS spectroscopyto samples flowing in microchannels. However, these results sufferedfrom severe limitations. In the study by Wang et al. [Wang et al.,Optics Express 16, 5782 (2008)], schematically represented in FIG. 1(a),an apparatus 100 includes a mode-locked laser oscillator 110 and amode-locked laser oscillator 120, a synchronization control module 130,a flowcell 140 containing a flow channel, and a microscope objective102. Mode-locked laser oscillators 110 and 120 are synchronized by acontrol signal 101 produced by synchronization control module 130;mode-locked laser oscillator 110 generates a pump pulse train 103 at awavelength corresponding to the desired CARS pump wavelength, andmode-locked laser oscillator 120 generates a Stokes pulse train 105 at awavelength corresponding to the desired CARS Stokes wavelength; the pumpand Stokes pulse trains are combined and focused by objective 102 ontoparticles for analysis in the flow channel of flowcell 140. Pulse trains103 and 105 are both a series of pulses which interact with a particlesuch that multiple pulses in the train interact with the particle. Noneof these pulses in these trains are chirped.

In this example, the CARS analysis is restricted to a single vibrationalCARS (anti-Stokes) frequency corresponding to the difference between theCARS pump frequency of pump pulse train 103 and the CARS Stokesfrequency of Stokes pulse train 105. Additionally, this apparatusachieves a sample flow velocity of about 40 mm/s—almost three orders ofmagnitude slower than a typical commercial flow cytometer and unsuitablefor laboratory-based analysis of real samples in a reasonable time.

In another example, the study by Camp Jr. et al. [Camp Jr. et al.,Optics Express 17, 22879 (2009)] schematically represented in FIG. 1(b),an apparatus 150 includes a mode-locked laser oscillator 160, a photoniccrystal fiber 170, a flowcell 180 containing a flow channel, and amicroscope objective 152. Mode-locked laser oscillator 160 generates apulse train 153 at a wavelength corresponding to the desired CARS pumpwavelength; a portion of such pulse train is split and fed into photoniccrystal fiber 170, while the rest continues as a pump pulse train 155.Photonic crystal fiber 170 generates by nonlinear optical processes aStokes pulse train 157 with light spread across a band of wavelengths,each acting as a CARS Stokes wavelength; the pump and Stokes pulsetrains are combined and focused by objective 152 onto particles foranalysis in the flow channel of flowcell 180. Pulse trains 153 and 155are both a series of pulses that interact with a particle such thatmultiple pulses in the train interact with the particle. None of thesepulses in these trains are chirped.

In this example, broader spectral analysis (referred to as multiplexCARS, or M-CARS) than that depicted in FIG. 1(a) is achieved through theuse of a photonic crystal fiber, a specialty optical fiber with anoptical damage threshold that poses severe constraints on the peak powerof the pulse train sent through it. As a result, this apparatus achievesa sample flow velocity of less than 200 μm/s—about 50,000 times slowerthan in conventional flow cytometers and unsuitable for laboratory-basedanalysis of real samples in a reasonable time. At these speeds, anybenefit accruing from broadband CARS spectroscopy is nullified by theresulting impractically low sample analysis throughput.

Existing solutions for analysis of microscopic particles thereforesuffer from a number of disadvantages, as follows:

dc impedance, ac impedance, optical extinction, and elastic lightscattering in flow cytometry; and brightfield, darkfield, phasecontrast, and other traditional optical techniques in microscopy, arenot of themselves sufficiently specific to produce information withwhich to reliably distinguish particles of different types, particularlyin many biological applications;

in flow cytometry and microscopy, use of standalone fluorophoresprovides limited specificity, and additionally requires incubation,which adds costs and reduces efficiency in high-throughput operations;

in flow cytometry and microscopy, use of fluorophore-conjugatedmonoclonal antibodies requires lengthy incubation, requires complex andexpensive measures for shipment and storage of the fragile reagents, andadds significant costs to operations on a per-assay basis;

in microscopy, whether traditional or coupled with advancedspectroscopic techniques like CARS, analysis of stationary samples ismuch too slow to practically, efficiently, and cost-effectively provideinformation on a sufficient numbers of particles to confer to suchanalysis the required statistical significance in most applications;

in CARS spectroscopy as demonstrated to analysis of flowing samples, thecurrent speed of analysis is much too slow for practical implementation,even when trade-offs are made in range of spectral coverage, resultingadditionally in very limited compositional information.

SUMMARY

Embodiments of apparatuses and methods to provide a label-free or nativeparticle analysis are described. In an embodiment, an apparatuscomprises a light generating system producing cross-chirped pairs offirst light pulses at a first wavelength and second light pulses at asecond wavelength; and optional flow cell (or other chamber) coupled tothe light generating system to convey or hold the particles foranalysis. An optical system is coupled to the light generating system tochirp at least one of the first light pulses and the second light pulsesto analyze the particles. The chamber can be similar to a microscopeslide if a flow cytometer is not used in one embodiment.

In an embodiment, an apparatus comprises a light generating systemproducing first light pulses at a first wavelength and second lightpulses at a second wavelength, the second wavelength being differentfrom the first wavelength; and a flow cell coupled to the lightgenerating system to convey particles for analysis. An optical system iscoupled to the light generating system to chirp at least one of thefirst light pulses and the second light pulses to analyze the particles.

In an embodiment, an apparatus comprises a light generating systemproducing first light pulses at a first wavelength and second lightpulses at a second wavelength; and a flow cell coupled to the lightgenerating system to convey particles for analysis. An optical system iscoupled to the light generating system to chirp at least one of thefirst light pulses and the second light pulses to analyze the particles.A collecting and analyzing system is coupled to the light generatingsystem to collect and analyze light resulting from an interaction of thefirst light pulses and the second light pulses with the particles.

In an embodiment, an apparatus comprises a light generating systemproducing first light pulses at a first wavelength and second lightpulses at a second wavelength; and a flow cell coupled to the lightgenerating system to convey particles for analysis. An optical system iscoupled to the light generating system to chirp at least one of thefirst light pulses and the second light pulses to analyze the particles.An optical module is coupled to the light generating system to combinethe first light pulses and the second light pulses to deliver to theparticles. The particles are each exposed to a single combined lightpulse from one of the first light pulses and one of the second lightpulses.

In an embodiment, an apparatus comprises a light generating systemproducing first light pulses at a first wavelength and second lightpulses at a second wavelength; and a flow cell coupled to the lightgenerating system to convey particles for analysis. An optical system iscoupled to the light generating system to chirp at least one of thefirst light pulses and the second light pulses to analyze the particles.A synchronization system is coupled to the light generating system tosynchronize the first light pulses and the second light pulses.

In an embodiment, an apparatus comprises a light generating systemproducing first light pulses at a first wavelength and second lightpulses at a second wavelength; and a flow cell coupled to the lightgenerating system to convey particles for analysis. An optical system iscoupled to the light generating system to chirp at least one of thefirst light pulses and the second light pulses to analyze the particles.A light source is coupled to the light generating system to illuminatethe particles to generate a trigger light.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing first light pulses at a first wavelength andsecond light pulses at a second wavelength. A flow cell is coupled tothe light generating system to convey a plurality of particles foranalysis. A first optical system is coupled to the light generatingsystem to generate combined light pulses from the first light pulses andthe second light pulses to deliver to the particles. The particles areeach exposed to one of the combined light pulses.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing first light pulses at a first wavelength andsecond light pulses at a second wavelength, the second wavelength beingdifferent from the first wavelength. A flow cell is coupled to the lightgenerating system to convey a plurality of particles for analysis. Afirst optical system is coupled to the light generating system togenerate combined light pulses from the first light pulses and thesecond light pulses to deliver to the particles. The particles are eachexposed to one of the combined light pulses.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing first light pulses at a first wavelength andsecond light pulses at a second wavelength. A flow cell is coupled tothe light generating system to convey a plurality of particles foranalysis. A first optical system is coupled to the light generatingsystem to generate combined light pulses from the first light pulses andthe second light pulses to deliver to the particles. The particles areeach exposed to one of the combined light pulses. A collecting andanalyzing system is coupled to the light generating system to collectand analyze light resulting from the interaction of the combined lightpulses with the particles.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing first light pulses at a first wavelength andsecond light pulses at a second wavelength. A flow cell is coupled tothe light generating system to convey a plurality of particles foranalysis. A first optical system is coupled to the light generatingsystem to generate combined light pulses from the first light pulses andthe second light pulses to deliver to the particles. The particles areeach exposed to one of the combined light pulses. A second opticalsystem is coupled to the light generating system to chirp at least oneof the first light pulses and the second light pulses.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing first light pulses at a first wavelength andsecond light pulses at a second wavelength. A flow cell is coupled tothe light generating system to convey a plurality of particles foranalysis. A first optical system is coupled to the light generatingsystem to generate combined light pulses from the first light pulses andthe second light pulses to deliver to the particles. The particles areeach exposed to one of the combined light pulses. A synchronizationsystem is coupled to the light generating system to synchronize thefirst light pulses and the second light pulses.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing a first light pulse at a first wavelengthand a second light pulse at a second wavelength. A flow cell coupled tothe light generating system to convey a particle for analysis. A firstoptical system coupled to the light generating system to generate acombined light pulse from the first light pulse and the second lightpulse to analyze the particle. A light source is coupled to the lightgenerating system to illuminate the particle to generate a triggeroptical beam for the combined light pulse.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing a first light pulse at a first wavelengthand a second light pulse at a second wavelength. A flow cell coupled tothe light generating system to convey a particle for analysis. A firstoptical system coupled to the light generating system to generate acombined light pulse from the first light pulse and the second lightpulse to analyze the particle. A light source is coupled to the lightgenerating system to illuminate the particle to generate a triggeroptical beam for the combined light pulse. A second optical systemcoupled to the light generating system to chirp at least one of thefirst light pulse and the second light pulse.

In an embodiment, a particle analysis apparatus comprises a lightgenerating system producing a first light pulse at a first wavelengthand a second light pulse at a second wavelength. A flow cell coupled tothe light generating system to convey a particle for analysis. A firstoptical system coupled to the light generating system to generate acombined light pulse from the first light pulse and the second lightpulse to analyze the particle. A light source is coupled to the lightgenerating system to illuminate the particle to generate a triggeroptical beam for the combined light pulse. A collecting and analyzingsystem is coupled to the light generating system to collect and analyzelight resulting from the interaction of the combined light pulse withthe particle.

In an embodiment, first light pulses are generated at a firstwavelength. Second light pulses are generated at a second wavelength.Particles are conveyed for analysis. At least one of the first lightpulses and the second light pulses is chirped to analyze the particles.

In an embodiment, first light pulses at a first wavelength aregenerated. Second light pulses at a second wavelength are generated.Particles are conveyed for analysis. Combined light pulses are generatedfrom the first light pulses and the second light pulses to deliver theparticles. Each particle is exposed to one of the combined light pulses.

In an embodiment, a first light pulse is generated at a firstwavelength. A second light pulse is generated at a second wavelength. Aparticle is conveyed for analysis. A combined light pulse is generatedfrom the first light pulse and the second light pulse to analyze theparticle. The particle is illuminated to generate a trigger optical beamfor the combined light pulse.

In an embodiment, a first light pulse at a first wavelength and a secondlight pulse at a second wavelength are generated. At least one of thefirst light pulse and the second light pulse are chirped to analyze aparticle. The first light pulse and the second light pulse are combinedto generate a combined chirped pulse. The particle is interrogated usingthe chirped combined pulse.

In accordance with at least one embodiment, an apparatus for label-freeparticle or native analysis comprises a channel for flow of particlessuspended in a carrier fluid, a laser system providing ultrafastamplified, synchronized, and cross-chirped pairs of light pulses, aspectrometer, a continuous-wave triggering light source, one or morelight scattering photodetectors, and an electronic detector and a dataprocessing system to collect, digitize, process, analyze, and presentthe signals from interactions between the laser light and particlespresented for analysis.

Advantages of one or more embodiments can include

a) a method and an apparatus for particle analysis that confercompositional specificity to the results on a particle-by-particlebasis, enabling effective differentiation of similar particles;

b) a method and an apparatus for particle analysis without the need forincubation in sample preparation;

c) a method and an apparatus for particle analysis without the need forreagents that are expensive, require complex and costly shipment andstorage procedures, and/or involve lengthy incubation steps;

d) a method and an apparatus for particle analysis that do not requirethe sample to be stationary;

e) a method and an apparatus for particle analysis that producespectroscopic compositional information on a particle-by-particle basisin flowing samples, with sufficient spectral coverage, and at highenough throughput to allow sufficient statistical analysis on largeensembles of particles.

Other features and advantages of embodiments will be apparent from theaccompanying drawings and from the detailed description that follows.

BRIEF DESCRIPTION OF DRAWINGS

The embodiments as described herein are illustrated by way of exampleand not limitation in the figures of the accompanying drawings in whichlike references indicate similar elements.

FIG. 1A illustrates a typical approach to CARS spectroscopy of flowingsamples.

FIG. 1B illustrates a typical approach to CARS spectroscopy of flowingsamples.

FIG. 2 is a schematic diagram illustrating an apparatus for particleanalysis in accordance with one embodiment.

FIG. 3A illustrates a spectroscopic method to be performed on theparticle analysis apparatus of FIG. 2 in accordance with one embodiment.

FIG. 3B illustrates a spectroscopic method to be performed on theparticle analysis apparatus of FIG. 2 in accordance with one embodiment.

FIG. 4A shows a graph illustrating relationships among spectralcharacteristics of certain items of FIG. 2 in accordance with oneembodiment.

FIG. 4B shows a graph illustrating relationships among spectralcharacteristics of certain items of FIG. 2 in accordance with oneembodiment.

FIG. 4C shows a graph illustrating relationships among spectralcharacteristics of certain items of FIG. 2 in accordance with oneembodiment.

FIG. 5A is a schematic representation of aspects of a sequence ofparticle interrogation events involving the apparatus of FIG. 2 inaccordance with one embodiment.

FIG. 5B is a schematic representation of aspects of a sequence ofparticle interrogation events involving the apparatus of FIG. 2 inaccordance with one embodiment.

FIG. 5C is a schematic representation of aspects of a sequence ofparticle interrogation events involving the apparatus of FIG. 2 inaccordance with one embodiment.

FIG. 6 is a perspective view of the particle interrogation region of theflow channel of FIG. 2 in accordance with one embodiment.

FIG. 7 is a flow chart describing a sequence of principal operationsinvolved in the performance of the method of particle analysis inaccordance with one embodiment.

FIG. 8 illustrates a spectroscopic method to be performed in accordancewith one embodiment.

FIG. 9 illustrates a spectroscopic method to be performed in accordancewith one embodiment.

FIG. 10 is a schematic representation of aspects of sample analysis inaccordance with one embodiment.

FIG. 11 shows a block diagram of an exemplary embodiment of a dataprocessing system to analyze particles as described herein.

DETAILED DESCRIPTION

The embodiments will be described with references to numerous detailsset forth below, and the accompanying drawings. The followingdescription and drawings are illustrative of the embodiments and are notto be construed as limiting. Numerous specific details are described toprovide a thorough understanding of the embodiments as described herein.However, in certain instances, well known or conventional details arenot described in order to not unnecessarily obscure the embodiments indetail.

Reference throughout the specification to “at least some embodiments”,“another embodiment”, or “an embodiment” means that a particularfeature, structure, or characteristic described in connection with theembodiment is included in at least some embodiments as described herein.Thus, the appearance of the phrases “in at least some embodiments” or“in an embodiment” in various places throughout the specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

In the description herein of the apparatus and its operation, the terms“beam” and “pulse” as referred to the output of pulsed lasers aresometimes used interchangeably. Both terms are accurate andcomplementary descriptions of pulsed laser outputs; when discussingspatial behavior, the term “beam” more fittingly captures the path thata laser output takes; while, when discussing temporal behavior, the term“pulse” is more appropriate. The term “pulse” will be understood to meanthat a light source is on (to illuminate a target) for a period of timeand is off (and hence not illuminating the target) for another period oftime. In an embodiment, a duration of the pulse is a period of timeduring which the beam is turned on (to illuminate a target).

Herein, the term “pulse beam” is sometimes used for the avoidance ofconfusion. It will be appreciated by someone skilled in the art thatthese various terms reflect different attributes of a single unifiedentity. Similarly, the terms “wavelength” (represented by “λ”),“wavenumber” (represented by “ω”) and “frequency” (represented by “ν”)of an optical pulse or beam, where ω=1/λ and ν=c/λ, c being the speed oflight, are used interchangeably to describe defining characteristics ofthe light in such pulse or beam. It will be further appreciated thatdepictions of elements in the figures provided are schematic and onlyindicative of relative sizes and configurations, the relative dimensionsof several elements having in places been altered from an embodiment forthe pursuit of illustrative clarity; that several elements desirable oreven necessary for the proper function of the apparatus, for theeffective performance of the method, or for both, are not shown in thefigures and in several cases are not explicitly mentioned in thisdescription, but would nevertheless be understood by someone skilled inthe art as forming implicit part of the apparatus, or contributingimplicitly to the method, or both, of the embodiments of the presentinvention.

DRAWINGS—REFERENCE NUMERALS

Element number Element name 100 apparatus of the prior art 101 controlsignal 102 microscope objective 103 pump pulse train 105 Stokes pulsetrain 110 mode-locked laser oscillator 120 mode-locked laser oscillator130 synchronization control module 140 flowcell 150 apparatus of theprior art 152 microscope objective 153 pulse train 155 pump pulse train157 Stokes pulse train 160 mode-locked laser oscillator 170 photoniccrystal fiber 180 flowcell 200 particle analysis apparatus 201 pulsebeam 202 beamsplitter 203 pulse beam 204 mirror 205 pulse beam 206dispersion control module 207 pulse beam 208 variable delay line 209Stokes pulse beam 210 ultrafast amplifier module 211 Stokes pulse beam212 mirror 213 Stokes pulse beam 214 dispersion control module 215 pulsebeam 216 dichroic mirror 217 pump pulse beam 218 beam shaper 219combined pump-Stokes pulse beam 220 OPA 221 combined pump-Stokes pulsebeam 222 dichroic mirror 223 combined pump-Stokes pulse beam 224 beamshaper 225 cw beam 226 microscope objective 227 cw beam 228 flow channel228′ flow channel 228″ flow channel 229 cw beam 230 cw source 231 cwbeam  231′ cw beam  231″ focused cw beam spot 232 microscope objective233 combined pump-Stokes pulse beam  233′ combined pump-Stokes pulsebeam  233″ focused combined pump-Stokes beam spot   233′″ combinedpump-Stokes pulse train 234 dichroic mirror 235 trigger beam  235′trigger beam 236 spectral filter 237 combined CARS pulse beam  237′combined CARS pulse beam  237″ combined CARS pulse train 238photodetector 239 trigger beam 240 flowcell  240′ flowcell  240″flowcell 241 trigger beam 242 dichroic mirror 243 trigger beam 244spectral filter 245 electrical trigger signal 246 spectral filter 247synchronization trigger signal 248 neutral density filter 249 electronictime stamp signal 250 spectrometer 251 combined CARS pulse beam 252photodetector 253 combined CARS pulse beam 255 anti-Stokes signal pulsebeam 257 anti-Stokes signal pulse beam 259 electrical gating signal 260photodetector array 261 coded anti-Stokes spectral signal 263 alteredcombined pump-Stokes pulse beam 265 altered pump pulse beam 267 alteredpump pulse electrical signal 269 digital packets 270 synchronizationcontrol module 280 data acquisition module 290 computer module 300frequency-time graph 301 Stokes center frequency 302 ellipse 303 Stokesbandwidth 305 pump center frequency 306 ellipse 307 pump bandwidth 309starting pulse duration 310 time axis 320 frequency axis 350frequency-time graph 352 Stokes ellipse 353 Stokes shift 356 pumpellipse 357 anti-Stokes shift 359 stretched pulse duration 363anti-Stokes center frequency 364 anti-Stokes ellipse 365 anti-Stokesbandwidth 402 cw wavelength 403 wavelength 404 anti-Stokes band 405wavelength 406 pump band 407 wavelength 408 Stokes band 410 wavelengthaxis 412 mark 413 spectral plot 414 mark 415 spectral plot 416 mark 417spectral plot 420 optical transmission axis 423 spectral plot 425spectral plot 427 spectral plot 500 particle interrogation region  500′particle interrogation region 502 top flowcell wall 504 bottom flowcellwall 506 sheath and carrier fluids  506′ sheath and carrier fluids 508vector 510 particle 512 first cw beam boundary 514 second cw beamboundary 516 first CARS beam boundary 518 second CARS beam boundary 602first flowcell sidewall 604 second flowcell sidewall 700 flow chart 702method operation 704 method operation 706 method operation 708 methodoperation 710 method operation 712 method operation 714 method operation716 method operation 718 method operation 720 method operation 722method operation 800 frequency-time graph 802 Stokes ellipse 806 pumpellipse 814 anti-Stokes ellipse 900 frequency-time graph 902 Stokesellipse 903 Stokes bandwidth 914 anti-Stokes ellipse 915 anti-Stokesbandwidth 1000  interrogation region 1006  fluid 1100  data processingsystem 1101  processing unit 1103  memory 1104  instructions and data1105  display controller 1107  display device 1109  non-volatile storage1111  I/O controller 1113  audio input device 1115  audio output 1117 I/O device 1119  digital image input device 1121  modem or networkinterface 1123  bus

An embodiment of the particle analysis apparatus is illustrated in FIG.2 (schematic diagram). Particle analysis apparatus 200 comprises anultrafast pulsed laser amplifier module (“ultrafast amplifier”) 210, anultrafast optical parametric amplifier module (“OPA”) 220, acontinuous-wave light source (“cw source”) 230, a flowcell 240(partially shown here), a spectrometer 250, a photodetector array 260, asynchronization control module (“sync”) 270, a data acquisition module(“DAQ”) 280, and a computer module (“PC”) 290. Ultrafast amplifier 210generates a pulse beam 201 that travels to a beamsplitter 202 and isseparated into a pulse beam 203 and a pulse beam 205. Pulse beam 205 isredirected by a mirror 204 into a pulse beam 207, which propagatesthrough a dispersion control module 206 and emerges as a Stokes pulsebeam 209. Stokes pulse beam 209 travels through a variable delay line208 and continues as a Stokes pulse beam 211 to a mirror 212, where itreflects into a Stokes pulse beam 213. Stokes pulse beam 213 transmitsthrough a dichroic mirror 216 as a combined pump-Stokes pulse beam 219.

Pulse beam 203 enters OPA 220, which generates a pulse beam 215. Pulsebeam 215 propagates through a dispersion control module 214 and emergesas a pump pulse beam 217, which is then reflected by dichroic mirror 216to combine and overlap with transmitted Stokes pulse beam 213 intocombined pump-Stokes pulse beam 219. In at least one embodiment, pumppulse beam 217 and Stokes pulse beam 213 are cross-chirped, as describedin further detail below. Combined pump-Stokes pulse beam 219 propagatesthrough a beam shaper 218 and continues as a combined pump-Stokes pulsebeam 221 to a dichroic mirror 222, where it is transmitted as a combinedpump-Stokes pulse beam 223.

Cw source 230 generates a cw beam 225, which propagates through a beamshaper 224 and continues as a cw beam 227 to dichroic mirror 222, whereit is reflected as a cw beam 229. Both cw beam 229 and combinedpump-Stokes pulse beam 223 travel through a microscope objective 226 toemerge, respectively, as cw beam 231 and combined pump-Stokes pulse beam233, and continue to propagate to flowcell 240. Flowcell 240 comprises aflow channel 228 (partially shown here) through which particles to beanalyzed move in fluid suspension. In other embodiments such as, e.g.,those in which a flow cytometer is not used, a sample holder (such as amicroscope slide or other container) holds a fixed, stationary sample.Cw beam 231 and combined pump-Stokes pulse beam 233 are focused bymicroscope objective 226 in flow channel 228. A more detaileddescription of the configuration of flow channel 228, of the focusedspots of beams 231 and 233, and of their interactions with flowingparticles is provided below in reference to FIGS. 5 and 6.

Additionally provided in apparatus 200, but not shown in FIG. 2 in theinterest of clarity, are: an extension of partially shown flowcell 240and partially shown flow channel 228, upstream of the shown portion,that include a hydrodynamic focusing portion for generation of aparticle-carrying carrier fluid forming a core sample stream within asheath fluid; a nozzle for introduction of the particle-carrying carrierfluid; a larger channel for introduction of the sheath fluid; anextension of partially shown flowcell 240 and partially shown flowchannel 228, downstream of the shown portion, that include connectionsand tubing for delivery of interrogated particles, carrier fluid, andsheath fluid to waste; and means of regulating the injection of both theparticle-carrying carrier fluid and the sheath fluid, at desiredrespective rates of volumetric flow, into said nozzle and larger channeland, through said hydrodynamic focusing portion, into the shown portionof flowcell 240 and flow channel 228. Design and incorporation of suchelements into apparatus 200 is well known in the art of flow cytometry.

The interaction of cw beam 231, as focused by microscope objective 226in flow channel 228, with flowing particles under analysis results in atrigger beam 235 that propagates out of flowcell 240, is collected by amicroscope objective 232, and emerges as a trigger beam 239, whichreflects off of a dichroic mirror 234 as a trigger beam 241, whichtravels through a spectral filter 236 and propagates as a trigger beam243 to a photodetector 238. Photodetector 238 converts the opticalsignal from trigger beam 243 into an electrical trigger signal 245,which is directed to data acquisition module 280, where it is recorded,and to synchronization control module 270. Synchronization controlmodule 270 generates a synchronization trigger signal 247 that isdirected to the synchronization and trigger circuitry of ultrafastamplifier module 210, and an electronic time-stamp signal 249 that isdirected to data acquisition module 280, where it is recorded.

The interaction of combined pump-Stokes pulse beam 233, as focused bymicroscope objective 226 into flow channel 228, with flowing particlesunder analysis results in a combined CARS pulse beam 237 that propagatesout of flowcell 240, is collected by microscope objective 232, andemerges as a combined CARS pulse beam 251, which transmits throughdichroic mirror 234 as a combined CARS pulse beam 253. Combined CARSpulse beam 253 hits a dichroic mirror 242, where it is separated into ananti-Stokes signal pulse beam 255 and an altered combined pump-Stokespulse beam 263. Anti-Stokes signal pulse beam 255 is directed through aspectral filter 244 and emerges as an anti-Stokes signal pulse beam 257,which enters spectrometer 250. Spectrometer 250 spectrally dispersesanti-Stokes signal pulse beam 257 for detection by a photodetector array260. Synchronization control module 270 generates an electrical gatingsignal 259 in response to electrical trigger signal 245, whichelectrical gating signal 259 is received by photodetector array 260. Inresponse to electrical gating signal 259, photodetector array 260converts the spectrally dispersed information from anti-Stokes signalpulse beam 257 into a coded anti-Stokes spectral signal 261 that istransmitted to data acquisition module 280, where it is recorded.

Altered combined pump-Stokes pulse beam 263 travels through a spectralfilter 246 that rejects its Stokes portion and transmits its pumpportion; the transmitted pump portion traverses a neutral-density filter248 and emerges as an altered pump pulse beam 265, which is received bya photodetector 252 and converted into an altered pump pulse electricalsignal 267, which is then transmitted to data acquisition module 280,where it is recorded. Data acquisition module 280 exchanges digitalpackets 269 (that include control signals, spectral data, and otherinformation) along an electronic communication bus with computer module290, where further signal processing occurs and where processed data isstored and displayed for the user to inspect, duplicate, or transfer toanother system for long-term storage. Computer module 290 furtherprovides an interface with which the user directs the apparatus toperform the particle analysis described herein.

Several elements of the apparatus of an embodiment of the presentinvention are not shown in FIG. 2 in the pursuit of clarity. Forexample, among the items not shown are: certain lenses for relay ofoptical beams; slits and pinholes for spatial filtering of opticalbeams; probes, syringes, syringe drives, pumps, valves, reservoirs, andtubing for the aspiration, staging, transference, storage, and deliveryof particle-containing samples to flowcell 240 for analysis and fordisposal of analyzed samples to waste; certain electrical cables for thetransmittance of power, control signals, and data signals; mechanicalsupports; housings; user interfaces for the control and interaction withthe apparatus. It will be apparent to someone skilled in the art howthese common elements integrate into the apparatus according to anembodiment of the present invention.

Operation

The method and apparatus of an embodiment of the present inventioninterrogates flowing particles on a single-shot basis (in oneembodiment), gathering a broad CARS spectrum from each interaction. Byenabling the analysis of an individual particle to occur in the span ofa single interrogation event by use of amplified ultrafast opticalpulses, an embodiment of the present invention overcomes theshortcomings of the prior art and allows the label-free analysis ofparticles in flowing samples at high throughput, providing detailedcomposition information on a particle-by-particle basis without the useof expensive reagents.

Time-Bandwidth Characteristics

Ultrafast amplifier module 210 is a pulsed laser module producingamplified ultrafast pulses of light, typically comprising an ultrafastpulsed laser oscillator, a pump laser for the oscillator, a multipassamplifier, a pump laser for the amplifier, and supporting elements suchas, e.g., electrical power supplies, air cooling subsystems, and controland synchronization electronics. A suitable multi-pass amplifier, forexample, is a laser amplifier known to those skilled in the art as aregenerative amplifier, based, e.g., on Chirped Pulse Amplification.

The components listed above of ultrafast amplifier module 210 may beseparate or they may be integrated into a single subsystem. Internallyto ultrafast amplifier module 210, the laser oscillator produces acontinuous train of ultrafast optical pulses at repetition rates ofabout tens of megahertz, each pulse typically being femtoseconds topicoseconds wide and having an energy in an approximate range of a fewto a few tens of microjoules. Synchronization trigger signal 247commands the synchronization and trigger circuitry of ultrafastamplifier module 210 to select individual pulses from the oscillatorpulse train, at rates in the Hz to MHz range, with which to seed themultipass amplifier; the multipass amplifier amplifies the energy insuch pulse by a factor of up to about a million, and the resultingamplified pulse is delivered from ultrafast amplifier module 210 aspulse beam 201.

OPA 220 is a nonlinear optical module capable of producing ultrafastoptical pulses at a wavelength tunable across a wide range of values,starting from an input ultrafast optical pulse at a given wavelength. Atypical OPA accepts near-infrared (NIR) pulses around 50 fs long with anenergy per pulse on the millijoule scale, and delivers output pulsessynchronized with the input pulses, having center wavelengths tunablefrom the ultraviolet (UV) to the mid-infrared (MIR), and having pulseenergies dependent on the tuning wavelength. The wavelength tuning ofOPA 220 is adjustable in the method according to an embodiment of thepresent invention. OPA 220 receives an individual pulse in pulse beam203 at a center wavelength determined by the narrow tuning parameters ofultrafast amplifier module 210, and it outputs an individual pulse inpulse beam 215 at a center wavelength determined by the broad tuningparameters of OPA 220, such output and input pulses being mutuallysynchronized.

FIGS. 3A and 3B are graphs that illustrate schematically thespectroscopic method according to an embodiment of the presentinvention. Referring to FIG. 3A and FIG. 2, a time (“t”) axis 310 and afrequency (“ν”) axis 320 are shown, defining a frequency-time graph 300advantageous to clarify the relationships of various pulse beams ofapparatus 200. In optics a relationship can be defined between theduration (“Δτ”) and the bandwidth (“Δν”) of any given pulse: the productΔτ Δν, commonly referred to as the time-bandwidth product, underordinary circumstances has a lower bound. A physical pulse thatsatisfies this lower bound condition is commonly called“transform-limited,” as its temporal and frequency profiles are Fouriertransforms of one another. When both the time profile and the frequencyprofile of such pulse are described by, e.g., Gaussian curves, the lowerbound of the time-bandwidth product of a transform-limited pulse assumesthe value of 0.44 (with time measured in seconds and frequency inHertz). A continuous range of values of both duration and bandwidth ofany given pulse exists that satisfies the lower-bound limit for thetime-bandwidth product. Physical pulses can experience either temporalbroadening or frequency broadening or both, resulting in time-bandwidthproducts larger than the lower bound applicable for the pulse profile inquestion. In the description herein of the spectroscopic methodaccording to an embodiment of the present invention, and particularly inreference to the frequency-time plots provided in FIGS. 3A and 3Bherein, graphical elements such as ellipses are used as mathematicalrepresentations of the frequency and temporal characteristics ofphysical entities such as pulses or pulse beams. Accordingly, the terms“pulse,” “beam,” “pulse beam,” and “ellipse” as referred to the outputof pulsed lasers are sometimes used interchangeably. It will beappreciated by someone skilled in the art that these various termsreflect different attributes of a single unified entity. Additionally,in describing characteristics associated with physical or mathematicalquantities having a Gaussian, or approximately Gaussian, profile (suchas, e.g., the time, frequency, or spatial variation of an ultrafastoptical pulse), it is often advantageous for the pursuit of clarity toadopt a convention to symbolically represent the profile in question bytwo points on either side of the maximum having equal intensity: forexample, the two points at the 50% level of intensity that are also usedto define the Full Width at Half Maximum (FWHM), or the two points atthe 1/e² level of intensity. In FIGS. 3A and 3B, 5A to 5C, and 6, pulsesare represented in such a way, with the time and frequencyrepresentations in FIGS. 3A and 3B using the FWHM convention and thespatial representations in FIGS. 5A to 5C and 6 using the 1/e²convention.

Referring to FIGS. 2 and 3A, under optimized operation of each ofultrafast amplifier module 210 and OPA 220, the time-bandwidth productfor each of pulse beams 201 and 215 can approach or achieve thetransform-limited lower bound. An ellipse 302 represents thefrequency-time characteristics of ultrafast amplifier module pulse beam201; the same ellipse, save for horizontal time offsets, represents aswell as the frequency-time characteristics of pulse beams 203, 205, and207, which are substantially unaffected in frequency characteristics bythe elements that separate them. An ellipse 306 represents thefrequency-time characteristics of OPA pulse beam 215. The relative timeoffset of pulse beam 215 with respect to pulse beam 201 due to internalpropagation in OPA 220 is not shown in FIG. 3A in the interest ofclarity. A starting pulse duration (“Δτ”) 309 is shown to beapproximately the same for both ellipse 302 and ellipse 306; a Stokesbandwidth (“Δν_(Stokes)”) 303 of ellipse 302 and a pump bandwidth(“Δν_(pump)”) 307 of ellipse 306 are shown to also have approximatelyequal value. Ellipse 302 is shown to have a Stokes center frequency(“<ν_(Stokes)>”) 301, and ellipse 306 is shown to have a pump centerfrequency (“<ν_(pump)>”) 305. Stokes center frequency 301 is shown to belower than pump center frequency 305 in accordance with pulse beam 201(as modified by subsequent elements) to act as the CARS pump beam andpulse beam 215 (as modified by subsequent elements) to act as the CARSStokes beam in the method according to an embodiment of the presentinvention.

Referring to FIG. 3B, a time (“t”) axis 310 and a frequency (“ν”) axis320 are shown, defining a frequency-time graph 350 analogous to thatdefined by axes 310 and 320 in FIG. 3A. Referring to FIGS. 3A, 3B, and2, propagation of pulse beams 207 and 215 through, respectively,dispersion control modules 206 and 214 in apparatus 200 according to anembodiment of the present invention causes changes in their respectivefrequency-time characteristics. Dispersion generally signifies thedifferent behavior experienced by beams, or portions of beams, havingdifferent optical frequencies (or, equivalently, wavelengths). In thiscontext, dispersion indicates the different propagation delaysexperienced by different frequency components of a pulse beam as suchpulse beam propagates through a given material. Accordingly, pulse beam215 (represented by ellipse 306) transits through dispersion controlmodule 214, which is chosen so as to impart positive dispersion to apulse beam transiting through it; the resulting pump pulse beam 217 isrepresented in FIG. 3B as a pump ellipse 356. A pulse of the kindrepresented by pump ellipse 356 is said to have positive “chirp,” wherechirp, in analogy to acoustics, indicates the progressive change infrequency content of an extended pulse as the pulse evolves in time:positive chirp indicates a progressive increase in frequency content ofa pulse over its duration. This temporal evolution can be traced in FIG.3B by going from left to right along pump ellipse 356: the frequency ofeach portion of the pulse that the ellipse represents shifts higher andhigher from the beginning (left) to the end (right) of the pulse(ellipse).

Pulse beam 201 (represented by ellipse 302) first undergoes tworeflections that do not significantly influence its frequencycharacteristics, and then transits through dispersion control module206, which is chosen so as to impart negative dispersion to a pulse beamtransiting through it; the resulting Stokes pulse beam 209 isrepresented in FIG. 3B as a Stokes ellipse 352. A pulse of the kindrepresented by Stokes ellipse 352 is said to have negative chirp: thefrequency of each portion of the pulse that the ellipse representsshifts lower and lower from the beginning (left) to the end (right) ofthe pulse (ellipse).

Propagation through dispersive control modules 206 and 214 produces anincrease in the temporal spread (“stretch”) of each of pulse beams 209and 217 with respect to the temporal spread of each of pulse beams 207and 215, respectively. Referring to FIGS. 3A and 3B, this increase isrepresented by a stretched pulse duration (“Δτ′”) 359, which is shown tobe approximately the same for both Stokes pulse beam 209 (represented byStokes ellipse 352) and pump pulse beam 217 (represented by pump ellipse356), and also considerably larger than starting pulse duration 309.

Neither the bandwidths nor the center frequencies of either pulse beam215 or pulse beam 207 are significantly affected by propagation throughdispersive elements. Accordingly, a Stokes center frequency <ν_(Stokes)>301 of Stokes ellipse 352 (Stokes pulse beam 209) in FIG. 3B is shown tobe approximately the same as Stokes center frequency <ν_(Stokes)> 301 ofellipse 302 (pulse beam 207) in FIG. 3A, and a pump center frequency<ν_(pump)> 305 of pump ellipse 356 (pump pulse beam 217) in FIG. 3B isshown to be approximately the same as pump center frequency <ν_(pump)>305 of ellipse 306 (pulse beam 215) in FIG. 3A. Similarly, a Stokesbandwidth Δν_(Stokes) 303 of Stokes ellipse 352 in FIG. 3B is shown tobe approximately the same as Stokes bandwidth Δν_(Stokes) 303 of ellipse302 in FIG. 3A, and a pump bandwidth Δν_(pump) 307 of pump ellipse 356in FIG. 3B is shown to be approximately the same as pump bandwidthΔν_(pump) 307 of ellipse 306 in FIG. 3A.

The relative time offset of Stokes pulse beam 209 as it emerges pastvariable delay line 208 as Stokes pulse beam 211 is not shown in theinterest of clarity. Likewise, the effect of subsequent propagation ofStokes pulse beam 213 and pump pulse beam 217 through possiblydispersive material, such as beam shaper 218, dichroic mirror 222,microscope objective 226, flowcell 240, and the sheath and carrierfluids in flow channel 228, is not shown in the interest of clarity. Thesetting of variable delay line 208 is adjusted during calibration ofapparatus 200 in order to cause optimal temporal overlap of the pump andStokes pulses in combined pump-Stokes pulse beam 233 at the point ofinterrogation in flow channel 228. This optimal temporal overlap isschematically represented in FIG. 3B by the vertical alignment ofellipses 352 and 356. Likewise, the overall dispersion of the pump andStokes beams through all the optical elements the two beams traverse intheir path to the sample is accounted for in the design and operation ofdispersion control modules 206 and 214, which are adjusted and set toresult in the desired frequency characteristics of each beam [asschematically represented in FIG. 3B] at the point of interrogation.

Referring to FIGS. 2 and 3B, the coherent interaction of the positivelychirped pump pulse beam 217 (ellipse 356) and the negatively chirpedStokes pulse beam 213 (ellipse 352) with material from particles underanalysis in flow channel 228 results in the generation of theanti-Stokes portion of combined CARS pulse beam 237. Chirping, and theattendant temporal stretching, of the pump and Stokes pulse beams tendsto minimize non-resonant background and other nonlinear factors (such assecond-harmonic generation) that would act to distort and mask the CARSsignal. In this embodiment, the anti-Stokes signal being collected isessentially co-propagating with both the pump pulse beam and the Stokespulse beam as combined CARS pulse beam 237. The anti-Stokes portion ofthe pulse beam that carries the desired CARS signal is separated fromthe rest of the CARS pulse beams as anti-Stokes signal pulse beam 257.An anti-Stokes ellipse 364 represents the frequency-time characteristicsof anti-Stokes signal pulse beam 257. The form of anti-Stokes ellipse364 indicates schematically, in one embodiment, the effect of the CARSprocess that generates the desired anti-Stokes signal: (i) stretchedpulse duration Δτ 359 of anti-Stokes ellipse 364 is shown to beapproximately the same as that of both Stokes ellipse 352 and pumpellipse 356; (ii) an anti-Stokes shift 357 between an anti-Stokes centerfrequency <ν_(anti-Stokes)> 363 of anti-Stokes ellipse 364 and pumpcenter frequency <ν_(pump)> 305 is shown to be approximately equal to aStokes shift 353 between pump center frequency <ν_(pump)> 305 and Stokescenter frequency <ν_(Stokes)> 301; and (iii) an anti-Stokes bandwidth(“Δν_(anti-Stokes)”) 365 of anti-Stokes ellipse 364 is shown to beapproximately equal to the sum of Stokes bandwidth Δν_(Stokes) 303 andtwice pump bandwidth Δν_(pump) 307 in accordance with the CARS process.

Dispersion control module 214 is designed to impart positive chirp topulse beam 215. Positive chirp is the effect of what is commonlyreferred to as normal dispersion in transparent media in the range ofwavelengths from the UV (ultraviolet) to the NIR (near infrared). Suchdispersion (characterized by a positive Group Velocity Dispersion, orGVD) causes higher-optical-frequency waves to travel slower through suchmedia, and therefore to emerge out of such media with longer delays,than waves with lower optical frequency. Accordingly, dispersion controlmodule 214 comprises a length of material transparent, and with a knowngroup velocity dispersion, over the wavelength range of pulse beam 215,such length and group velocity dispersion being chosen so as to producethe desired degree of chirp and temporal stretching in pump pulse beam217.

Dispersion control module 206 is designed to impart negative chirp topulse beam 207. To achieve negative chirp, dispersion control module 206comprises dispersive elements arranged so as to cause an overallanomalous dispersion through dispersion control module 206. Suchdispersion (characterized by a negative Group Velocity Dispersion, orGVD) causes higher-optical-frequency waves to travel faster through suchmodule, and therefore to emerge out of such media with shorter delays,than waves with lower optical frequency. Devices generally known aspulse compressors, comprising, e.g., sets of matched dispersive prismsarranged to produce negative chirp by angular dispersion, are well knownin the art, as are other arrangements of dispersive elements such as,e.g., matched grating pairs. Such devices are generally used in theprior art to impart negative chirp to compensate for unwanted positivechirp and therefore achieve maximally compressed pulses. In the methodand apparatus according to an embodiment, devices of such design areused for the opposite purpose, i.e., to stretch short pulses byimparting a negative chirp. Accordingly, dispersion control module 206comprises a set of matched dispersive elements transparent over thewavelength range of pulse beam 207 arranged to produce, by angulardispersion, the desired degree of negative chirp and temporal stretchingin Stokes pulse beam 209.

Spectral Characteristics

FIGS. 4A to 4C are graphs that illustrate schematically the spectralrelationships among several elements of particle analysis apparatus 200according to an embodiment. Referring to FIG. 4A, several spectral bandsare shown along a wavelength (“λ”) axis 410 to indicate theirapproximate relative values. Starting with lower wavelength values atthe left, one encounters first a cw wavelength 402, representing thewavelength of cw source 230; an anti-Stokes band 404, representing thewavelength range of anti-Stokes signal pulse beam 257; a pump band 406,representing the wavelength range of pump pulse beam 217 (and also ofpulse beam 215); and a Stokes band 408, representing the wavelengthrange of Stokes pulse beams 209, 211, and 213 (and also of pulse beams201, 205, and 207). A wavelength 403 is shown, between cw wavelength 402and anti-Stokes band 404, chosen so as not to overlap with either cwwavelength 402 or anti-Stokes band 404. Likewise, a wavelength 405 isshown, between anti-Stokes band 404 and pump band 406, chosen so as notto overlap with either anti-Stokes band 404 or pump band 406. Likewise,a wavelength 407 is shown, between pump band 406 and Stokes band 408,chosen so as not to overlap with either pump band 406 or Stokes band408. Wavelengths 403, 405, and 407 are shown also in FIGS. 4A to 4C tofacilitate comparison.

FIG. 4B is a schematic graph of the spectral characteristics of severaldichroic elements of particle analysis apparatus 200, plotted on anoptical transmission (“T”) axis 420 vs. a wavelength (“λ”) axis 410.Axis 410 in FIG. 4B is analogous to axis 410 in FIG. 4A, and the twoaxes are mutually aligned to illustrate the relationships between thewavelength bands of FIG. 4A and the spectral characteristics of FIG. 4B.Axis 420 bears a mark 412 indicating the value of 0%, representing zerotransmission; a mark 414 indicating the value of 50%, representing 50%transmission; and a mark 416 indicating the value of 100%, representing100% transmission. In optical transmission graphs of the kindillustrated in FIG. 4B, as long as absorption and scattering losses ofthe plotted elements are negligible, one can also extract values ofoptical reflection as the complement of optical transmission: therefore,a point showing transmission of 0% also indicates reflection of 100%;one showing transmission of 50% also indicates reflection of 50%; andone showing transmission of 100% also indicates reflection of 0%; andsimilarly for all points in between 0% and 100%.

Starting from the lower wavelength values at the left, one encountersfirst a spectral plot 413, representing the optical transmission of bothdichroic mirror 222 and dichroic mirror 234; then a spectral plot 415,representing the optical transmission of dichroic mirror 242; and aspectral plot 417, representing the optical transmission of dichroicmirror 216. Each of spectral plots 413, 415, and 417 shows a transition,going from short to long wavelengths, from a behavior of very lowtransmission and very high reflection to a behavior of very hightransmission and very low reflection. This type of spectral behavior iscommonly referred to as “longpass” on account of the fact that longerwavelengths are passed through the corresponding element and shorterwavelengths are reflected by it.

The wavelength where each plot crosses the T=50% level is called theedge wavelength (“λ_(edge)”) of the corresponding dichroic mirror: Theλ_(edge) of the dichroic mirrors represented by plot 413 matcheswavelength 403, the λ_(edge) of the mirror represented by plot 415matches wavelength 405, and the λ_(edge) of the mirror represented byplot 417 matches wavelength 407. The edge wavelengths of the dichroicmirrors in the apparatus according to an embodiment of the presentinvention are chosen so as to respond differently to appropriatewavelength bands and act either as beam combiners or as beam splitters,depending on the configuration.

Accordingly, the λ_(edge) shown by wavelength 403 is chosen so that thebehavior indicated by plot 413 is to reflect cw wavelength 402 andtransmit anti-Stokes band 404, pump band 406, and Stokes band 408.Therefore, in the configuration shown in FIG. 2, dichroic mirror 222acts as a beam combiner and dichroic mirror 234 acts as a beam splitterfor the sets of wavelengths indicated. Similarly, the λ_(edge) shown bywavelength 405 is chosen so that the behavior indicated by plot 415 isto reflect anti-Stokes band 404 and transmit pump band 406 and Stokesband 408. Dichroic mirror 242 acts therefore as a beam splitter, in theconfiguration shown in FIG. 2, for the sets of wavelengths indicated.And the λ_(edge) shown by wavelength 407 is chosen so that the behaviorindicated by plot 415 is to reflect pump band 406 and transmit Stokesband 408. Dichroic mirror 216 acts therefore as a beam combiner, in theconfiguration shown in FIG. 2, for the sets of wavelengths indicated.

FIG. 4C is a schematic graph of the spectral characteristics of severalfilter elements of particle analysis apparatus 200, plotted on opticaltransmission (“T”) axis 420 vs. a wavelength (“λ”) axis 410. Axis 410 inFIG. 4C is analogous to axis 410 in FIG. 4A and axis 410 in FIG. 4B, andthe three axes are mutually aligned to illustrate the relationshipsbetween the wavelength bands of FIG. 4A and the spectral characteristicsof FIGS. 4B and 4C. Axis 420 bears a mark 412 indicating the value of0%, representing zero transmission; a mark 414 indicating the value of50%, representing 50% transmission; and a mark 416 indicating the valueof 100%, representing 100% transmission. Representation of opticaltransmission and optical reflection in the graph of FIG. 4C is analogousto that in the graph of FIG. 4B. Starting from the lower wavelengthvalues at the left, one encounters first a spectral plot 423,representing the optical transmission of filter 236; then a spectralplot 425, representing the optical transmission of filter 244; and aspectral plot 427, representing the optical transmission of filter 246.Each of these plots shows a transition, going from short to longwavelengths, from a behavior of very high transmission and very lowreflection to a behavior of very low transmission and of very highreflection. This type of spectral behavior is commonly referred to as“shortpass” on account of the fact that shorter wavelengths are passedthrough the corresponding element and longer wavelengths are reflectedby it.

The wavelength where each plot crosses the T=50% level is called theedge wavelength λ_(edge) of the corresponding filter: The λ_(edge) ofthe filter represented by plot 423 matches wavelength 403, the λ_(edge)of the filter represented by plot 425 matches wavelength 405, and theλ_(edge) of the filter represented by plot 427 matches wavelength 407.The edge wavelengths of the filters in the apparatus according to anembodiment of the present invention are chosen so as to increase thedegree of rejection of undesired wavelength bands.

Accordingly, the λ_(edge) shown by wavelength 403 is chosen so that thebehavior of filter 236 indicated by plot 423 is to transmit and acceptcw wavelength 402 and reflect and reject anti-Stokes band 404, pump band406, and Stokes band 408. Similarly, the λ_(edge) shown by wavelength405 is chosen so that the behavior of filter 244 indicated by plot 425is to transmit and accept anti-Stokes band 404 and reflect and rejectpump band 406 and Stokes band 408. And the λ_(edge) shown by wavelength407 is chosen so that the behavior of filter 246 shown by plot 427 is totransmit and accept pump band 406 and reflect and reject Stokes band408.

Cw source 230 is designed to produce a beam to be used as a trigger forultrafast amplifier module 210 and OPA 220 of particle analysisapparatus 200. Referring to FIGS. 2 and 4A, cw wavelength 402 isdesigned to be shorter than the shortest wavelength in anti-Stokes band404. This design requirement translates in practice in ensuring asufficient spectral gap between cw wavelength 402 and anti-Stokes band404 so that a reasonably manufacturable long-pass dichroic mirror 234will transmit a majority of anti-Stokes band 404 of the light incombined CARS pulse beam 251 while reflecting a majority of cwwavelength 402 in trigger beam 239. The spectral cutoff profiles ofcommercially available longpass dichroic filters and the range ofwavelengths of commercially available cw sources are sufficient toensure that this is the case.

Photodetector 238 is chosen to have an appropriately sensitive responseat cw wavelength 402, and a sufficiently fast response to produce anelectrical signal that closely approximates the actual temporalvariation of optical signal 243 incident upon it. Integrated intophotodetector 238, or adjunct to it, is signal-amplifying circuitry thatboosts the level of electrical trigger signal 245 in a predominantlylinear proportional relationship to the level of optical signal 243received by photodetector 238.

Beamsplitter 202 is designed to have an essentially flat spectralresponse across Stokes band 408 of pulse beam 201. It is furtherdesigned to transmit a majority of the light incident upon it as pulsebeam 203, and reflect the balance as pulse beam 205. The energy perpulse of OPA output pulse beam 215 depends on the specific wavelengthOPA 220 is tuned to. Accordingly, as the intent is generally forcombined pump-Stokes pulse beam 233 to deliver approximately comparablepulse energies in the Stokes pulse and in the pump pulse to theparticles under analysis, the transmission/reflection ratio forbeamsplitter 202 is chosen in support of that goal, taking inconsideration the efficiency of light conversion taking place in OPA 220at the chosen tuning wavelength.

Mirrors 204 and 212 are designed to have an essentially flat spectralresponse across Stokes band 408 of pulse beam 203 and Stokes pulse beam211. The internal mirrors that comprise variable delay line 208 arelikewise designed to have a similarly flat response across Stokes band408.

Neutral-density filter 248 is designed to have an essentially flatspectral response across pump band 406 and, therefore, of the portion ofaltered combined pump-Stokes pulse beam 263 that is transmitted throughfilter 246. Neutral-density filter 248 is further designed to have asufficient optical density, or opacity, to reduce the peak power ofaltered pump pulse beam 265 to levels below both the optical damagethreshold and the limit of linear response of photodetector 252.

Photodetector 252 is chosen to have an appropriately sensitive spectralresponse to pump band 406, and a time response as fast as practical toproduce an electrical signal that introduces the least distortion intothe actual temporal variation of optical signal 265 incident upon it.Integrated into photodetector 252, or adjunct to it, issignal-amplifying circuitry that boosts the level of altered pump pulseelectrical signal 267 in a predominantly linear proportionalrelationship to the level of optical signal 265 received byphotodetector 252.

Spectrometer 250 is designed to spectrally disperse the wavelengthcomponents of anti-Stokes signal pulse beam 257 in such a way as topermit their detection and resolution by photodetector array 260.Spectrometer 250 may consist of gratings, prisms, or other spectrallydispersive elements, and it may consist of a single stage, a doublestage, or more, as appropriate to provide the desired spectralresolution without unduly sacrificing sensitivity.

Photodetector array 260 may be, e.g., a CCD camera, a CMOS camera, asensor array, a photomultiplier array, or any other suitable devicecapable of detecting with sufficient sensitivity light levels along acollection of discrete positions disposed in a direction aligned withthe direction of spectral dispersion by spectrometer 250. Photodetectorarray 260 is chosen to have an appropriately sensitive spectral responseto anti-Stokes band 404 generated by the interaction of combinedpump-Stokes pulse beam 233 with particles under analysis. Photodetector260 may further be designed to allow for cooling of its active sensorelements in order to reduce background electrical noise. Photodetectorarray 260 is further designed to allow for activation and deactivationof its sensing function by gating signal 259 on a timescale ascomparable as possible to the duration of any single pulse inanti-Stokes signal pulse beam 257.

Geometry and Temporal Characteristics

FIGS. 5A to 5C are side-view cross-sectional schematic representationsof the particle interrogation region in the apparatus according to anembodiment of the present invention that illustrate the relativeconfigurations of the interrogating beams in relationship to the flowchannel and particles therein, at three instances of time depicting theprogression of a particle under analysis within the flow channel.Referring to FIG. 5A, a particle interrogation region 500 is showncomprising portion of flowcell 240 from FIG. 2 shown here as a flowcell240′ and portion of flow channel 228 from FIG. 2 shown here as a flowchannel 228′ within flowcell 240′, both flowcell and flow channel beingshown as sectioned along a vertical midplane that contains both thecentral axis of the flow channel and the central axes of theinterrogating beams. Further shown in FIG. 5A are portion of a topflowcell wall 502, portion of a bottom flowcell wall 504, portion ofsheath and carrier fluids 506, a vector 508 indicating direction andvelocity of fluid flow and of particle motion, portion of cw beam 231from FIG. 2 shown here as a cw beam 231′, a first cw beam boundary 512of cw beam 231′, a second cw beam boundary 514 of cw beam 231′, and aparticle 510 at a time when its position in flow channel 228′ isupstream of cw beam 231′. Referring to FIGS. 5B and 2, particleinterrogation region 500 is shown at a later time when particle 510 hasadvanced to a position of overlap with cw beam 231′, the interactionwith which transiently generates trigger beam 235, portion of which isshown here as a trigger beam 235′. Referring to FIG. 5C, particleinterrogation region 500 is shown, additionally comprising portion ofcombined pump-Stokes pulse beam 233 from FIG. 2 shown here as a combinedpump-Stokes pulse beam 233′, a first CARS beam boundary 516 of combinedpump-Stokes pulse beam 233′, and a second CARS beam boundary 518 ofcombined pump-Stokes pulse beam 233′, at a time later yet when particle510 has further advanced to a position of overlap with combinedpump-Stokes pulse beam 233′, the interaction of which transientlygenerates combined CARS pulse beam 237, portion of which is shown hereas a combined CARS pulse beam 237′. Depictions of elements in thecross-sectional views of FIGS. 5A to 5C are schematic and onlyindicative of relative sizes and configurations, the relative dimensionsof several elements being illustrated differently from an embodiment forthe pursuit of illustrative clarity. For example, particle 510 is shownas only slightly smaller than the height of flow channel 228′, whereasin practice it would be advantageous for particle 510 to be considerablysmaller than the height of flow channel 228′. Also in the way ofexample, the sheath fluid and the particle-carrying carrier fluidforming a core sample stream within the sheath fluid are not separatelydemarcated in FIGS. 5A to 5C.

In FIGS. 5A to 5C, cw beam 231′ and combined pump-Stokes pulse beam 233′are schematically represented by boundaries, respectively 512 and 514,and 516 and 518. These boundaries represent idealizations of the waistof the beam in each case, and specifically the locations in eachhorizontal plane of the 1/e² levels of intensity in the respectivespatial profiles, following common practice in Gaussian optics and theconvention outlined above with respect to representations of Gaussianprofiles. In practice, the intensity profiles of real beams havegenerally more gradual transition than the indication of a boundarymight suggest. Accordingly, the extent of each beam in practice reachesbeyond the boundaries schematically indicated, although generally withdecreasing intensity.

Cw beam 231′ is shown present at all the times represented in FIGS. 5Ato 5C, reflecting the fact that it is a beam from a cw source. On theother hand, combined pump-Stokes pulse beam 233′ is shown only presentat the time represented in FIG. 5C, reflecting the fact that it is abeam from an ultrafast pulsed source. Pulse beam 233′ is only generatedin response to the transit of particles through the interrogation regionas indicated schematically by the sequence illustrating the process forparticle 510 in FIGS. 5A to 5C, and it is specifically triggered by thepassing of such particles through the region illuminated by cw beam231′.

FIG. 6 is a perspective view of a cross-sectional schematicrepresentation of the particle interrogation region of flow channel 228to illustrate the relative configurations of the interrogating beams inrelationship to the flow channel and particles therein. Referring toFIG. 6, a view of particle interrogation region 500 from FIGS. 5A to 5Cis shown here as a particle interrogation region 500′, comprising a viewof flowcell 240′ from FIGS. 5A to 5C shown here as a flowcell 240″, aview of flow channel 228′ from FIGS. 5A to 5C shown here as a flowchannel 228″ within flowcell 240″, both flowcell and flow channel beingshown as sectioned along a horizontal midplane that contains the centralaxis of the flow channel and is approximately perpendicular to thepropagation direction of the interrogating beams. Further shown in FIG.6 are a first flowcell sidewall 602, a second flowcell sidewall 604,sheath and carrier fluids 506′, vector 508 indicating direction andvelocity of fluid flow and of particle motion, portion of cw beam 231from FIG. 2 shown here as a focused cw beam spot 231″, portion ofcombined pump-Stokes pulse beam 233 from FIG. 2 shown here as a focusedcombined pump-Stokes beam spot 233″, and particle 510 in a position ofoverlap with focused combined pump-Stokes beam spot 233″ correspondingto the time represented also in FIG. 5C.

Depictions of elements in the cross-sectional view of FIG. 6 areschematic and only indicative of relative sizes and configurations, thepresence and position of several elements being illustrated differentlyfrom an embodiment for the pursuit of illustrative clarity. For example,focused cw beam spot 231″ and focused combined pump-Stokes beam spot233″ are shown as only slightly smaller than the width of flow channel228″, whereas in practice it would be advantageous for both beam spotsto be considerably smaller than the width of flow channel 228′. Also inthe way of example, the sheath fluid and the particle-carrying carrierfluid forming a core sample stream within the sheath fluid are notseparately demarcated in FIG. 6. Further, focused cw beam spot 231″ andfocused combined pump-Stokes beam spot 233″ are schematicallyrepresented by ellipses. These ellipses represent idealizations of thewaist of the beam in each case, and specifically the locations, in thefocal plane of microscope objective 226, of the 1/e² levels of intensityin the respective spatial profiles, following common practice inGaussian optics and the convention outlined herein with respect torepresentations of Gaussian profiles. In practice, the intensityprofiles of real beams have generally more gradual transition than theindication of a line boundary might suggest. Accordingly, the extent ofeach beam in practice reaches beyond the ellipses schematicallyindicated, although generally with decreasing intensity.

Beam shaper 218 shapes combined pump-Stokes pulse beam 219 in such a wayas to achieve, in conjunction with microscope objective 226, the desiredoptical properties for focused combined pump-Stokes beam spot 233″ inthe focal plane of microscope objective 226 in flow channel 228. Outputpulse beams 201 and 215 from, respectively, ultrafast amplifier module210 and OPA 220 generally are collimated, have approximately Gaussianprofiles, and have circular or elliptical cross sections. Beam shaper218 is designed, by selection and placement of such appropriaterefractive or reflective elements as may comprise it (such as, e.g.,diverging and converging lenses or mirrors; where both lenses or mirrorsmay be spherical to affect equally both cross-sectional dimensions ofthe beam; or both lenses or mirrors may be cylindrical, to affect onecross-sectional dimension of the beam but not the other), to altercombined pump-Stokes pulse beam 219 and produce optical characteristics(including, e.g., dimensions, a cross-sectional aspect ratio, and adivergence) of combined pump-Stokes pulse beam 221 to result, in thefocal plane of microscope objective 226 in flow channel 228, in afocused combined pump-Stokes beam spot 233″ with desired opticalcharacteristics (such as, e.g., dimensions, a cross-sectional aspectratio, and a divergence in at least one dimension in a desired range ofvalues in order to satisfy as broadly as possible wavevector sum rulesfor CARS processes).

Beam shaper 224 shapes cw beam 225 in such a way as to achieve, inconjunction with microscope objective 226, the desired opticalproperties for focused cw beam spot 231″ in the focal plane ofmicroscope objective 226 in flow channel 228. Output cw beam 225 from cwsource 230 is generally chosen to be collimated, have an approximatelyGaussian profile, and have a circular or elliptical cross section. Beamshaper 224 is designed, by selection and placement of appropriaterefractive or reflective elements as may comprise it (such as, e.g.,diverging and converging lenses or mirrors; where both lenses or mirrorsmay be spherical to affect equally both cross-sectional dimensions ofthe beam; or both lenses or mirrors may be cylindrical, to affect onecross-sectional dimension of the beam but not the other), to alter cwbeam 225 and produce optical characteristics (including, e.g.,dimensions, a cross-sectional aspect ratio, and a divergence) of cw beam227 to result, in the focal plane of microscope objective 226 in flowchannel 228, in a focused cw beam spot 231″ with desired opticalcharacteristics (such as, e.g., dimensions, a cross-sectional aspectratio, and a divergence in a desired range of values in order to conferto cw beam 231′ a sufficient depth of focus).

Microscope objective 226 is designed to focus combined pump-Stokes pulsebeam 223 and cw beam 229 onto particles under analysis in the focalplane of microscope objective 226, approximately in the midplane of flowchannel 228, and to produce the desired dimensions of focused combinedpump-Stokes pulse beam spot 233″ and focused cw beam spot 231″. The beamfocusing geometry is calculated to satisfy with high tolerance the CARSwavevector sum rules in the plane defined by the propagation axis ofcombined pump-Stokes beam 233′ and vector 508. A suitable choice ofmicroscope objective 226 is an air-gap, long-working-distance,infinity-corrected achromatic objective.

Microscope objective 232 is designed to collect the greatest portion ofcombined CARS pulse beam 237′ generated in the course of the interactionbetween combined pump-Stokes pulse beam 233′ and particle 510, and torelay the light thus collected to spectrometer 250 through theintervening optical elements. Microscope objective 232 is secondarilydesigned to also collect a sufficient portion of trigger beam 235 toallow for the reliable detection of particles flowing in flow channel228 and subsequent triggering of ultrafast amplifier module 210 and OPA220. A suitable choice of microscope objective 232 is an air-gap,long-working-distance, infinity-corrected achromatic objective.

Referring to FIGS. 2, 5A to 5C, and 6, a typical sequence of events isdescribed comprising interrogation of particles under analysis in themethod according to an embodiment of the present invention. At the timeof FIG. 5A, representative particle 510 is yet to reach the region whereinteraction with any of the laser beams is to take place. At the time ofFIG. 5B, particle 510 has reached the region of interaction with cw beam231′; the result of that interaction, represented by trigger beam 235′,is used as a trigger signal that ultimately results in combinedpump-Stokes pulse beam 233′ interrogating particle 510 at the time ofFIG. 5C. The time interval t_(b-c) that elapses between the time of FIG.5B and that of FIG. 5C depends on the distance d_(b-c) between focusedcw beam spot 231″ and focused combined pump-Stokes beam spot 233″ in thefocal plane of microscope objective 226 in flow channel 228″, and on thevelocity ν_(particle) of particle flow in the flow channel. Both suchdistance d_(b-c) and such velocity ν_(particle) can be adjusted within arange of practically achievable values; their design values are set toresult in a time interval t_(b-c) sufficiently long to allow for thepropagation of optical and electrical signals through the apparatus. Forthe purpose of adjusting ν_(particle), the effective rate of injectionof sheath and carrier fluids 506 into flow channel 228″ is adjusted. Forthe purpose of adjusting d_(b-c), dichroic mirror 222 is aligned andoriented to direct cw beam 229 through microscope objective 226 and intoflowcell 240″ in such a way as to place focused cw beam spot 231″upstream (with respect to the direction of fluid flow in flow channel228″ shown by vector 508) from focused combined pump-Stokes beam spot233″. Distance d_(b-c) is designed to be such that the time intervalt_(b-c) it takes a particle to traverse it closely approximates thecumulative time it takes for a signal (optical or electrical asrespectively relevant) to propagate along the path comprising segments235, 239, 241, 243, 245, 247, 201, 205, 207, 209, 211, 213, 219, 221,223, and 233, plus any significant internal delay in interveningcomponents, including 504, 232, 236, 238, 260, 210, 206, 208, 216, 218,222, 226, and 502. Synchronization control module 270 is used duringoperation to calibrate such interval t_(b-c) and make any necessaryresidual adjustments in the timely generation of synchronization triggersignal 247, to so that combined pump-Stokes pulse beam 233′ arrives atthe flow channel at the time particle 510 is passing through the regionwhere such pulse will intersect it.

Synchronization system 270 comprises sufficiently fast electroniccircuitry with sufficiently low timing noise, or jitter, to enablefaithful generation of electrical gating signal 259, synchronizationtrigger signal 247, and electronic time stamp signal 249 in response toelectrical trigger signal 245 without introducing unacceptable amountsof temporal spread or distortion.

Data acquisition module 280 is a multichannel digitization and signalprocessing module. It is set up to acquire input signals fromphotodetector 238, photodetector 252, synchronization control module270, and photodetector array 260, and to exchange control signals andprocessed data with computer module 290. Photodetector array 260produces coded anti-Stokes spectral signal 261 at a rate generallylimited by the maximum repetition rate of ultrafast amplifier module210. Coded anti-Stokes spectral signal 261 is acquired by dataacquisition module 280 and digitally associated therein with electricaltrigger signal 245, altered pump pulse electrical signal 267, andelectronic time stamp signal 249, all of these signals being mutuallysynchronized up to a fixed and calibratable time offset. Dataacquisition module 280 digitizes analog input waveforms at a sufficientdigitization frequency to faithfully record signal variations from therespective sources, performs signal buffering, and associates into adiscrete data packet all of the signals pertaining to an individualparticle analysis event, further transmitting one such data packet perparticle analysis event to computer module 290.

Computer module 290 provides a user interface with which to controluser-adjustable parameters of operation of particle analysis apparatus200 according to the method according to an embodiment of the presentinvention, and to direct such apparatus to perform a particle analysisaccording to such method. Computer module 290 further provides means toprocess and store particle analysis data transferred to it by dataacquisition module 280, and means to optionally view, analyze, andtransfer such data to long-term storage systems. Spectral informationobtained on apparatus 200 using the method in accordance with anembodiment of the present invention may be analyzed in various waysknown in the art, such as, e.g., by comparison, differencing, overlay,or otherwise correlative inspection of recorded spectra from particlesunder analysis with one or more spectra of known substances orparticles; by quantification of individual spectral features, such as,e.g., the presence, or height over background, or width, or shape, ofone or more spectral peaks; by principal component analysis; or by othersuch spectral analysis procedures.

Referring to FIG. 7, a flow chart 700 is provided that describes asequence of principal operations involved in the performance of themethod of particle analysis in accordance with an embodiment of thepresent invention. At a method operation 702, a user or operator ofapparatus 200 introduces into apparatus 200 a sample containing aplurality of particles for analysis; apparatus 200 aspirates, aliquots,or otherwise isolates a portion or a totality of such sample for opticalinterrogation; such portion or totality is injected, as aparticle-carrying carrier fluid and in conjunction with the simultaneousinjection of sheath fluid, into flow channel 228 within flowcell 240,thereby forming a core sample stream bounded by sheath fluid andprogressing in the direction of vector 508; such core sample streamcarries particles to interrogation region 500 where they are presentedfor optical interrogation.

At a method operation 704, a particle 510 of the plurality of particlesin the sample enters cw beam 231′ with wavelength 402; a transientoptical signal in the form of trigger beam 235′ is generated as a resultof the passage of particle 510 in cw beam 231′; such transient opticalsignal is collected by microscope objective 232, reflects off dichroicmirror 234, is isolated by undesired light of other wavelengths byfilter 236, and is converted by photodetector 238 into electricaltrigger signal 245; electrical trigger signal 245 is received bysynchronization control module 270 and used to generate synchronizationtrigger signal 247 and electronic time stamp signal 249.

At a method operation 706, ultrafast amplifier module 210 receivessynchronization trigger signal 247; timing electronic circuitry andcontrol firmware internal to ultrafast amplifier module 210 verifywhether ultrafast amplifier module 210 is ready to produce the desiredultrafast output pulse, based on the minimum pulse-repetition period ofultrafast amplifier module 210; if the result of such verification isnegative, the process is repeated starting with method operation 704; ifthe result is positive, a next method operation 708 is taken.

At method operation 708, ultrafast amplifier module 210 generates asingle pulse with Stokes band 408; beamsplitter 202 transmits part ofthe intensity of such pulse to OPA 220 and reflects the rest throughmirror 204 to dispersion control module 206; OPA 220 generates a singlepulse with pump band 406 synchronized to the pulse from ultrafastamplifier 210.

At a method operation 710, the pulse from OPA 220 is positively chirpedas ellipse 356 by dispersion control module 214 and sent as a pump pulseof pump pulse beam 217 to dichroic mirror 216; the pulse from ultrafastamplifier 210 is negatively chirped as ellipse 352 by dispersion controlmodule 206, delayed by variable delay line 208, and reflected by mirror212 as a Stokes pulse of Stokes pulse beam 213 to dichroic mirror 216,where it combines with the pump pulse in time and space as a combinedpulse of combined pump-Stokes pulse beam 219; the combined pulse isaltered in spatial dimensions by beam shaper 218, passes throughdichroic mirror 222, enters microscope objective 226, and is focusedthrough top flowcell wall 502 and through portion of sheath and carrierfluids 506 into flow channel 228′ as a combined pulse of combinedpump-Stokes pulse beam 233′.

At a method operation 712, the combined pulse interrogates particle 510by optical processes comprising inelastic light scattering, such as,e.g., Coherent anti-Stokes Raman Scattering (CARS); an anti-Stokes lightsignal is generated as ellipse 364 with anti-Stokes band 404, theforward-propagating portion of which combines with the interrogatingcombined cross-chirped pulse pair to form a combined CARS pulse ofcombined CARS pulse beam 237′.

At a method operation 714, the combined CARS pulse propagates throughportion of sheath and carrier fluids 506, propagates through bottomflowcell wall 504, is collected by microscope objective 232, and passesthrough dichroic mirror 234 to reach dichroic mirror 242; the combinedCARS pulse is then separated into the anti-Stokes signal portion, whichis reflected through spectral filter 244 to spectrometer 250, and thecombined pump-Stokes portion, which reaches spectral filter 246; theStokes portion of the combined pump-Stokes portion is rejected byspectral filter 246; the pump portion is transmitted by spectral filter246, attenuated by neutral-density filter 248, and reaches photodetector252.

At a method operation 716, synchronization control module 270 generateselectrical gating signal 259 and transmits it to photodetector array260; the anti-Stokes signal is dispersed by spectrometer 250 into ananti-Stokes spectrum transmitted to photodetector array 260;photodetector array 260 integrates the optical spectrum incident upon itfor the duration of electrical gating signal 259, which is timed tosynchronize with the anti-Stokes signal; photodetector array 260converts the anti-Stokes spectrum received from spectrometer 250 intocoded anti-Stokes spectral signal 261; photodetector 252 converts thepump portion of the combined CARS pulse into altered pump pulseelectrical signal 267.

At a method operation 718, data acquisition module 280 receives, atvarious time delays with respect to the passage of particle 510 throughcw beam 231′, electrical trigger signal 245, electronic time stampsignal 249, coded anti-Stokes spectral signal 261, and altered pumppulse electrical signal 267; data acquisition module 280 converts anyanalog signals received into digitized equivalents; data acquisitionmodule 280 optionally processes such received and digitized signals toimprove their signal-to-noise characteristics; data acquisition module280 provides for a temporary storage of the received and digitizedsignals as associated items in a digital packet identified by its timestamp on an onboard memory module; data acquisition module 280optionally transmits such digital packet to computer module 290 forfurther preliminary processing.

At a method operation 720, a firmware function determines, on the basisof user-defined parameters and default parameters (such as, for exampleand without limitation, the number of individual particle spectracollected from the sample being analyzed; the number such spectrasatisfying predetermined criteria; the time elapsed since the sample wasintroduced; the interruption by the user or operator), whether apparatus200 should be directed to analyze yet more particles; if the result ofsuch determination is positive, the analysis process is repeatedstarting with method operation 704; if the result is positive, a nextmethod operation 722 is taken. At method operation 720, another firmwarefunction periodically directs apparatus 200 to collect backgroundspectra from the carrier fluids in the absence of particles for use inbackground subtraction and other signal processing functions.

At method operation 722, digital packets corresponding to each recordedparticle-interrogation event since the sample was introduced foranalysis, plus digital packets corresponding to background spectra, aretransmitted from data acquisition module 280 to computer module 290;computer module 290 performs digital signal processing and data analysisfunctions on such packets, such as, for example without limitation,background subtraction, smoothing, upsampling, Fourier filtering, noisefiltering, baseline subtraction, peak detection, normalization,averaging, weighted averaging, signal differencing, comparison tolibraries, comparison to look-up tables, feature extraction, principalcomponent analysis, support vector machine analysis, Bayesian analysis,and/or other spectral processing and analysis functions as are known inthe art; computer module 290 further stores the results of suchprocessing and analysis, individually and/or in aggregate; computermodule 290 optionally presents such results to the user or operator viaa user interface, or optionally transmits such results to a separateunit for long-term storage, aggregation, and/or further analysis.

In an embodiment, the trigger functions (trigger beam, trigger detector,electrical trigger signal, synchronization trigger signal) are notneeded to analyze particles the concentration of which is substantiallyhigh. The combined CARS pulses are generated periodically to interrogatethe particle as described for example, in operation 712, and areunsynchronized with the passage of particles in the flow channel.Synchronization is retained between the combined CARS pulses and theelectrical gating signal for the photodetector array. The volumetricconcentration of particles in the core sample stream is arranged to besufficiently high so that each combined CARS pulse has a correspondinglysufficiently high probability of interacting with a particle in thesample stream. For a pulse repetition rate of, for example, 5 kHz, andan interaction probability of, for example, 10%, the effective resultingaverage event interaction is 500 particles per second.

In another embodiment, the trigger functions (trigger beam, triggerdetector, electrical trigger signal, synchronization trigger signal) arenot needed to analyze fluids (such as, e.g., pure fluids, mixtures,solutions, suspensions, multi-phase fluids, etc.), as described infurther detail below. For example, the fluid may be a solution of one ormore solute analyte compounds dissolved in a solvent. The combined CARSpulses are generated periodically to interrogate fluids in a mannersimilar to that described, for example, in operation 712, and arefree-running with respect to the passage of fluid(s) in the flowchannel. Synchronization is retained between the combined CARS pulsesand the electrical gating signal for the photodetector array. Eachcombined CARS pulse interrogates a volumetric portion of the fluid(s)presented for analysis, and the collection of multiple spectra from aplurality of combined CARS pulses yields an overall assay of thefluid(s) presented for analysis. The spectral signals resulting from theinteraction of the combined pulse with the fluid(s) are separated fromthe background noise, collected, and processed, for example as describedwith respect to operation 716. For a pulse repetition rate of, forexample, 1 kHz, 1,000 CARS spectra per second are acquired from thefluid(s) under analysis.

In another embodiment, the trigger functions (trigger beam, triggerdetector, electrical trigger signal, synchronization trigger signal) arenot needed, and laser oscillators replace the laser amplifiers, toanalyze fluids (such as, e.g., pure fluids, solutions, mixtures,suspensions, multi-phase fluids, etc.), as described in further detailbelow. For example, the fluid may be a solution of one or more soluteanalyte compounds dissolved in a solvent. The combined CARS pulses fromtwo laser oscillators operating at different wavelengths are generatedperiodically to interrogate fluids in a manner similar to thatdescribed, for example, in operation 712, and are free-running withrespect to the passage of fluid(s) in the flow channel. Each combinedCARS pulse interrogates a volumetric portion of the fluid(s) presentedfor analysis, and the accumulation of multiple spectra from a pluralityof combined CARS pulses yields an overall analysis of the fluid(s)presented for analysis. The signals resulting from the interaction ofthe combined pulse with the fluid(s) are separated from the backgroundnoise, collected, and processed, for example as described with respectto operation 716. The photodetector array is optionally arranged tointegrate multiple CARS spectra and generate an aggregate signal. For apulse repetition rate of, for example, 100 MHz, and an integration timeof 1 ms, 1,000 integrated spectra per second are recorded, each spectrumintegrating approximately 100,000 individual interactions betweencombined CARS pulses and portions of the fluid(s) under analysis.

Examples

An example of the embodiment with values of the main parameters ofoperation is provided herein. For simplicity of illustration, threeseparate selections of wavelength tuning (“Selection A”, “Selection B”,and “Selection C”) are offered as possible instances, withoutlimitation, of different settings for the same apparatus according to anembodiment. Referring to Table 1, for each column corresponding to adifferent wavelength tuning selection, center wavelength (<λ>, in nm),corresponding center wavenumber (<ω>, in cm⁻¹), pulse duration (Δτ, infs), and bandwidth (Δω, in cm⁻¹) are each shown for pulse beam 201 fromultrafast amplifier module 210 (underscript “amp”); for Stokes pulsebeam 209 after chirping by dispersion control module 206 (underscript“Stokes”); for pulse beam 215 from OPA 220 (underscript “OPA”); and forpump pulse beam 217 after chirping by dispersion control module 214(underscript “pump”). Table 1 additionally shows center wavelength andcorresponding center wavenumber for anti-Stokes signal pulse beam 257(underscript “anti-Stokes”); and the approximate range of Ramantransitions (indicated by the low 50% point, Ω_(Raman) low, and the high50% point, Ω_(Raman) high) accessible for each wavelength tuningselection. Quantities such as Δτ and Δω indicate the approximateFull-Width at Half-Maximum (FWHM) values of the respective temporal andwavenumber profiles of the pulses they describe. Likewise, Ω_(Raman) lowand Ω_(Raman) high indicate the two points approximately at half-maximumof the anti-Stokes bandwidth profile.

TABLE 1 Example of operating parameters with three choices of OPA tuningwavelength. Quantity Selection A Selection B Selection C units <λ_(amp)>800 800 800 nm <ω_(amp)> 12,500 12,500 12,500 cm⁻¹ Δτ_(amp) 40 40 40 fsΔω_(amp) 367 367 367 cm⁻¹ <λ_(Stokes)> 800 800 800 nm <ω_(Stokes)>12,500 12,500 12,500 cm⁻¹ Δτ′_(Stokes) 5 5 5 ps Δω_(Stokes) 367 367 367cm⁻¹ <λ_(OPA)> 733 685 643 nm <ω_(OPA)> 13,643 14,599 15,552 cm⁻¹Δτ_(OPA) 40 40 40 fs Δω_(OPA) 367 367 367 cm⁻¹ <λ_(pump)> 733 685 643 nm<ω_(pump)> 13,643 14,599 15,552 cm⁻¹ Δτ′_(pump) 5 5 5 ps Δω_(pump) 367367 367 cm⁻¹ <λ_(anti-Stokes)> 676 599 538 nm <ω_(anti-Stokes)> 14,78516,697 18,604 cm⁻¹ Ω_(Raman) low 593 1,549 2,502 cm⁻¹ Ω_(Raman) high1,693 2,649 3,602 cm⁻¹

It will be noted that, insofar as the parameters reported in Table 1 areconcerned, the only difference between the characteristics of pulse beam“amp” and pulse beam “Stokes” is the temporal width, due to chirping.Likewise, the only difference between the characteristics of pulse beam“OPA” and pulse beam “pump” is the temporal width, again due tochirping. Accordingly, the bandwidth shown for pulse beams “amp” and“OPA” is the transform-limited value corresponding to their pulsedurations; whereas the bandwidth shown for pulse beams “Stokes” and“pump” is the bandwidth each of them inherits from pulse beams “amp” and“OPA,” respectively.

In this example of the embodiment, ultrafast amplifier module 210comprises an ultrafast oscillator and a Chirped PulseAmplification-based Ti:sapphire regenerative amplifier tuned to a centerwavelength of approximately 800 nm, as well as the required pump lasersand supporting components. A commercially available laser systemsuitable for use as ultrafast amplifier module 210 is the Libra-HE+ USPmodel (Coherent, Santa Clara, Calif., USA). Ultrafast amplifier module210 is set to produce output pulses at a rate dictated by the arrival ofparticles for analysis, with an upper bound given by the maximumrepetition rate of ultrafast amplifier module 210 (such repetition rateis, for example, in the approximate range of 1 Hz to 1 MHz, in theapproximate range of 100 Hz to 100 kHz, and in more specific embodiment,in the approximate range of 1 to 10 kHz). The energy per pulse for pulsebeam 201 from ultrafast amplifier module 210 is, for example, in theapproximate range of 1 μJ to 100 mJ, in the approximate range of 100 μJto 25 mJ, and in more specific embodiment, in the approximate range of 1mJ to 5 mJ, with beamsplitter 202 selecting approximately 90% of thepulse energy for transmission as pulse beam 203 and reflecting theremaining 10% as pulse beam 205. OPA 220 is set to deliver the secondharmonic of the nonlinear frequency conversion signal at a centerwavelength, given an input pulse beam 203 at a wavelength of 800 nm,generally tunable from below 600 nm to about 800 nm. (Other choices ofsignal output from OPA 220 are possible, resulting in tuning overdifferent wavelength ranges both above and below the wavelength of inputpulse beam 203.) A commercially available optical parametric amplifiersystem suitable for use as OPA 220 is the TOPAS model (Coherent, SantaClara, Calif., USA). Referring to Table 1, for wavelength tuningselection A, OPA 220 is tuned to a center wavelength of about 733 nm,resulting in an energy per pulse for pulse beam 215 of about 160 μJ(assuming a 3.5 mJ input pulse at 800 nm); for selection B, OPA 220center wavelength is about 685 nm and the pulse energy about 220 μJ; forselection C, OPA 220 center wavelength is about 643 nm and the pulseenergy about 240 μJ. The bandwidth of the “amp,” “Stokes,” “OPA,” and“pump” pulses is approximately the same and equal to approximately 367cm⁻¹. The bandwidth of the “anti-Stokes” pulse is the result of the CARSfrequency-mixing process and is approximately equal to 1,100 cm⁻¹.

With the operating parameter choices indicated in Table 1, the apparatusaccording to an embodiment of the present invention is capable ofperforming particle analysis by single-shot CARS spectroscopy over abroad range of Raman transitions. With wavelength tuning selection A,the approximate range of Raman transitions covered is 600 to 1700 cm⁻¹;with selection B, the approximate range is 1,550 to 2,650 cm⁻¹; withselection C, the approximate range is 2,500 to 3,600 cm⁻¹. Selection Acovers the region of the Raman spectrum commonly known as thefingerprint region; selection C covers the region of the Raman spectrumfeaturing prominent lipid and water peaks; and selection B covers theregion of the spectrum lying in between the fingerprint region and thelipid/water peaks. The spectral range for selection B partially overlapswith the spectral ranges of both selection A and selection C tofacilitate spectral calibration across the combined range ofapproximately 600 to approximately 3,600 cm⁻¹.

In this example of the embodiment, cw source 230 produces a beam with acenter wavelength of approximately 488 nm and a power in the approximaterange of 1 to 100 mW, and in more specific embodiment, in theapproximate range of 5 to 25 mW. The cross-sectional dimension of flowchannel 228 in the direction parallel to light propagation is, forexample, in the approximate range of 50 to 1000 μm, in the approximaterange of 100 to 500 μm, and in more specific embodiment in theapproximate range of 200 to 300 μm. The cross-sectional dimension offlow channel 228 in the direction perpendicular to light propagation is,for example, in the approximate range of 50 to 1000 μm, in theapproximate range of 100 to 500 μm, and in more specific embodiment, inthe approximate range of 200 to 300 μm. The velocity of carrier fluidand particles in the center of the channel along vector 508 is, forexample, in the approximate range of 0.01 to 50 m/s, in the approximaterange of 0.1 to 20 m/s, and in more specific embodiment, in the range of1 to 10 m/s. The approximate dimensions of focused cw beam spot 231″ are10 μm in the direction of vector 508 and 80 μm in the directionperpendicular to vector 508. The approximate dimensions of focusedcombined pump-Stokes beam spot 233″ are 0.6 μm in the direction ofvector 508 and 10 μm in the direction perpendicular to vector 508.Particles to be analyzed are, for example, in the approximate range of50 nm to 50 μm, in the approximate range of 500 nm to 20 μm, and in morespecific embodiment, in the approximate range of 1 to 10 μm. Electricalgating signal 259 has a duration greater than the duration of stretchedpulse duration Δτ′ and, for example less than 100 μs, less than 100 ns,and in more specific embodiment, less than 500 ps; and is synchronizedto bracket stretched pulse duration Δτ′. Spectrometer 250 has, forexample an approximate spectral resolution of better than about 20 cm⁻¹,better than about 10 cm⁻¹, and in more specific embodiment, better thanabout 5 cm⁻¹.

It will be apparent from the foregoing description that the method andapparatus of embodiments of the present invention offer means to carryout particle analyses rapidly, with sensitivity to chemical composition,over ensembles of large numbers of particles, and without the use ofexpensive reagents. While the foregoing description contains manyspecificities, these should not be construed as limitations on the scopeof the invention, but rather as an exemplification of one embodimentthereof. Many other variations are possible. For example:

-   -   1) An alternative embodiment where variable delay line is        removed from the path between pulse beams 209 and 211 and        inserted instead in the path of pulse beam 217 in order to        satisfy the requirement for temporal overlap of pump and Stokes        pulse beams given a specific beam path geometry, component        layout, and internal delays. An alternative embodiment where OPA        220 is tuned to a wavelength longer than that produced by        ultrafast amplifier 210; where the roles of ultrafast amplifier        module 210 and OPA 220 are thereby reversed in the CARS process,        i.e., pulse beam 217 acts as the Stokes beam and pulse beam 213        acts as the pump beam; and where longpass dichroic mirror 216 is        replaced by a shortpass dichroic mirror with an edge wavelength        in between the center wavelengths of ultrafast amplifier 210 and        OPA 220.    -   2) An alternative embodiment where ultrafast amplifier module        210 comprises any of a fixed-wavelength fiber-based ultrafast        pulsed laser oscillator, a fixed-wavelength free-space ultrafast        pulsed laser oscillator, a fiber-based pump laser for the        oscillator, a fixed-wavelength fiber-based amplifier, a        fixed-wavelength free-space amplifier, a fiber-based pump laser        for the amplifier, a single-pass amplifier stage, a multi-pass        amplifier stage, or any suitable combination thereof.    -   3) Referring to FIGS. 2 and 8, an alternative embodiment where        dispersion control module 206 is designed to impart a positive        chirp, e.g., by comprising a length of material with positive        Group Velocity Dispersion; where the resulting Stokes pulse beam        209 is represented in FIG. 8 on frequency-time graph 800 as a        Stokes ellipse 802; where dispersion control module 214 is        designed to impart a negative chirp, e.g., by comprising a pulse        compressor arrangement of matched prisms or matched gratings        with overall anomalous dispersion characteristics; where the        resulting pump beam 217 is represented in FIG. 8 as a pump        ellipse 806; and where the anti-Stokes portion of combined CARS        pulse beam 237 is represented in FIG. 8 as an anti-Stokes        ellipse 814. An alternative embodiment where settings internal        to ultrafast amplifier module 210 are used to generate a        negatively chirped pulse with the desired characteristics, i.e.,        a temporally stretched pulse; and where dispersion control        module 206 is removed.    -   4) An alternative embodiment where settings internal to OPA 220        are used to generate a positively chirped pulse with the desired        characteristics, i.e., a temporally stretched pulse; and where        dispersion control module 214 is removed.    -   5) An alternative embodiment where OPA 220 is replaced by a        second ultrafast amplifier module, e.g., a module similar to        ultrafast amplifier 210; where both ultrafast amplifier module        210 and such second module produce, or are tuned to, wavelengths        suitable for performing the method according to an embodiment of        the present invention; and where ultrafast amplifier module 210        and such second module are mutually synchronized by means of        synchronization electronics including but not necessarily        limited to synchronization control module 270.    -   6) Referring to FIGS. 2 and 9, an alternative embodiment        incorporating the changes of alternative embodiment (5); where,        additionally, dispersion control module 206 is removed; where        ultrafast amplifier module 210 is set to produce        transform-limited picosecond-duration pulses; where the        resulting Stokes pulse beam 209 is represented in FIG. 9 on        frequency-time graph 900 as a Stokes ellipse 902; and where the        anti-Stokes portion of combined CARS pulse beam 237 is        represented in FIG. 9 as an anti-Stokes ellipse 914 having        anti-Stokes bandwidth Δν_(anti-stokes) 915. An alternative        embodiment where longpass dichroic mirrors 216, 222, 234, 242,        or any combination thereof, are replaced by shortpass analogues        with similar respective edge wavelengths; and where the geometry        of affected beam paths and the layout of affected components are        adjusted accordingly.    -   7) An alternative embodiment where shortpass filters 236, 244,        246, or any combination thereof, are supplemented by bandpass        filters with acceptable transmission values over the respective        wavelengths or wavelength bands 402, 404, and 406 and acceptable        extinction values over other wavelength ranges containing        undesired signals.    -   8) An alternative embodiment incorporating the changes of        alternative embodiment (7); and where, additionally, shortpass        filters 236, 244, 246, or any combination thereof, are removed.    -   9) An alternative embodiment where shortpass filter 236 is        supplemented by a notch filter with high extinction over band        406 and with acceptable transmission at wavelength 402, a notch        filter with high extinction over band 408 and with acceptable        transmission at wavelength 402, or both such notch filters.    -   10) An alternative embodiment where shortpass filter 244 is        supplemented by a notch filter with high extinction at        wavelength 402 and with acceptable transmission over band 404, a        notch filter with high extinction over band 406 and with        acceptable transmission over band 404, a notch filter with high        extinction over band 408 and with acceptable transmission over        band 404, or any combination thereof.    -   11) An alternative embodiment where shortpass filter 246 is        supplemented by a notch filter with high extinction at        wavelength 402 and with acceptable transmission over band 406, a        notch filter with high extinction over band 408 and with        acceptable transmission over band 406, or both such notch        filters.    -   12) An alternative embodiment where cw source 230 is directed        onto the particles under analysis in flow channel 228 by means        of an optical path propagating separately from combined        pump-Stokes pulse beams 223 and 233 and not passing through        microscope objectives 226 and 232; where an additional focusing        lens is placed between beam shaper 224 and flowcell 240 to        obtain the desired focused cw beam spot dimensions; where an        additional collection lens is placed between flowcell 240 and        spectral filter 236; where dichroic mirrors 222 and 234 are        removed; and where the beam path from cw source 230 to        photodetector 238 proceeds through beam shaper 224, said        additional focusing lens, flowcell 240, flow channel 228, said        additional collection lens, and spectral filter 236.    -   13) An alternative embodiment where cw source 230 comprises a        laser with an output wavelength of value within the approximate        range of 200 nm to 5000 nm; and where such wavelength is        sufficiently shorter than the shortest wavelength in anti-Stokes        band 404 to prevent interference with or from the CARS signal.    -   14) An alternative embodiment where cw source 230 comprises an        LED, a superluminescent LED, or other incoherent light source,        optionally suitably filtered, with an output band having a        center wavelength within the approximate range of 200 nm to 5000        nm; and where the longest wavelength in such output band is        sufficiently shorter than the shortest wavelength in anti-Stokes        band 404 to prevent interference with or from the CARS signal.    -   15) An alternative embodiment where longpass dichroic mirrors        222 and 234 are replaced by shortpass dichroic mirrors having an        edge wavelength longer than the longest wavelength in Stokes        band 408; where shortpass spectral filter 236 is replaced by a        longpass spectral filter with a similar edge wavelength to that        of said shortpass dichroic mirrors; where cw source 230        comprises a laser with an output wavelength of value within the        approximate range of 355 nm to 1700 nm; and where the wavelength        of cw source 230 is sufficiently longer than the edge wavelength        of said shortpass dichroic mirrors to prevent interference with        or from the Stokes beam.    -   16) An alternative embodiment where longpass dichroic mirrors        222 and 234 are replaced by shortpass dichroic mirrors having an        edge wavelength longer than the longest wavelength in Stokes        band 408; where shortpass spectral filter 236 is replaced by a        longpass spectral filter with a similar edge wavelength to that        of said shortpass dichroic mirrors; where cw source 230        comprises an LED, a superluminescent LED, or other incoherent        light source, optionally suitably filtered, with an output band        having a center wavelength within the approximate range of 200        nm to 5000 nm; and where the shortest wavelength of cw source        230 is sufficiently longer than the edge wavelength of said        shortpass dichroic mirrors to prevent interference with or from        the Stokes beam.    -   17) An alternative embodiment where beam shaper 218 is designed        to produce, in conjunction with microscope objective 226, a        focused combined pump-Stokes beam spot 233″ with approximately        equal dimensions in the directions parallel to and perpendicular        to vector 508; and where such dimensions are, for example, in        the approximate range of 0.2 to 20 μm, in the approximate range        of 0.5 to 10 μm, and in more specific embodiment, in the        approximate range of 1 to 5 μm.    -   18) An alternative embodiment where dichroic mirror 242 is        removed from its location between dichroic mirror 234 and        spectral filter 246, and placed in the path of beam 221 between        beam shaper 218 and dichroic mirror 222; where such repositioned        dichroic mirror 242 is oriented to reflect light below its edge        wavelength 405 away from beam 221 and toward an accessible        direction; where spectral filter 244 and spectrometer 250 with        associated photodetector array 260 are suitably repositioned to        receive light reflected by such repositioned dichroic mirror        242; and where any anti-Stokes signal light backpropagating        along beam 221 from the interaction region in flowcell 240 is        thusly reflected by such repositioned dichroic mirror 242,        filtered by such repositioned spectral filter 244, and measured        by repositioned spectrometer 250 and photodetector array 260.    -   19) Referring to FIGS. 2, 5(c) and 10, an alternative embodiment        where the following elements are removed: 230, 224, 222, 234,        236, 238, 246, 248, 252; where ultrafast amplifier module 210        and OPA 220 are each replaced by a mode-locked ultrafast laser        oscillator; where each such mode-locked ultrafast laser        oscillator is tuned to a wavelength suitable for performing the        method according to an embodiment of the present invention;        where each such mode-locked ultrafast laser oscillator produces        a continuous train of transform-limited,        femtosecond-scale-duration, nanojoule-scale-energy pulses at        repetition rates, for example, in the approximate range of 1 MHz        to 1 GHz, and, in more specific embodiment, in the approximate        range of 10 MHz to 100 MHz; where such mode-locked ultrafast        laser oscillators are mutually synchronized by means of        synchronization electronics including but not necessarily        limited to synchronization control module 270; where, referring        to an interrogation region 1000 in FIG. 10, sheath fluid and        particle-carrying carrier fluid 506 are replaced by a fluid 1006        under analysis; where fluid 1006 may comprise, e.g., a single        fluid of known or unknown composition, a solution with one or        more unknown solutes, a solution with an unknown concentration        of one or more known solutes, a mixture of one or more unknown        fluids, a mixture with an unknown concentration of one or more        known fluids, a suspension with one or more populations of        unknown solid components, a suspension with an unknown        concentration of one or more known solid components, a        multi-phase medium (including, but not limited to, emulsions and        aerosols) with two or more unknown phases, a multi-phase medium        (including, but not limited to, emulsions and aerosols) with an        unknown ratio of two or more known phases, or any combination        thereof; where a combined pump-Stokes pulse train 233′″ is        designed to focus inside flow channel 228′, interrogate a volume        of fluid 1006, and produce by such interrogation a combined CARS        pulse train 237″; where and where electrical gating signal 259        is modified to permit accumulation and integration in        photodetector array 260 of dispersed spectra from a plurality of        individual interrogation events.

It will be apparent to someone skilled in the art that many of theforegoing alternative embodiments may be usefully combined to produceyet other distinct alternative embodiments. For example, alternativeembodiments (3), (3), (4), and (5) may be combined to result in anotheralternative embodiment comprising two ultrafast amplifier modules, notcomprising either dispersion control module 206 or dispersion controlmodule 214, and capable of performing the spectroscopic methodillustrated in FIG. 8. Accordingly, the scope of the invention should bedetermined not by the embodiments illustrated, but by the appendedclaims and their legal equivalents.

FIG. 11 shows a block diagram of an exemplary embodiment of a dataprocessing system 1100 to provide a label-free or native particleanalysis as described herein. In an embodiment, data processing system1100 is a part of the control system to perform a method that includesgenerating first light pulses at a first wavelength; generating secondlight pulses at a second wavelength; conveyed particles for analysis,where least one of the first light pulses and the second light pulses ischirped to analyze the particles, as described herein. In an embodiment,data processing system 1100 is represented by a computer module 290depicted in FIG. 2.

Data processing system 1100 includes a processing unit 1101 that mayinclude a microprocessor or microprocessor, such as Intel microprocessor(e.g., Core i7, Core 2 Duo, Core 2 Quad, Atom), Sun Microsystemsmicroprocessor (e.g., SPARC), IBM microprocessor (e.g., IBM 750),Motorola microprocessor (e.g., Motorola 68000), Advanced Micro Devices(“AMD”) microprocessor, Texas Instrument microcontroller, and any othermicroprocessor or microcontroller.

Processing unit 1101 may include a personal computer (PC), such as aMacintosh® (from Apple Inc. of Cupertino, Calif.), Windows®-based PC(from Microsoft Corporation of Redmond, Wash.), or one of a wide varietyof hardware platforms that run the UNIX operating system or otheroperating systems. For at least some embodiments, processing unit 1101includes a general purpose or specific purpose data processing systembased on Intel, AMD, Motorola, IBM, Sun Microsystems, IBM processorfamilies, or any other processor families. As shown in FIG. 11, a memory1103 is coupled to the processing unit 1101 by a bus 1123. Memory 1103has instructions and data 1104 stored thereon which when accessed byprocessing unit 1101 cause the processing unit 1101 to perform methodsto provide label free or native particle analysis, as described herein.

Memory 1103 can be dynamic random access memory (“DRAM”) and can alsoinclude static random access memory (“SRAM”). A bus 1123 couplesprocessing unit 1101 to memory 1103 and also to a non-volatile storage1109 and to a display controller 1105 (if a display is used) and to aninput/output (I/O) controller(s) 1111. Display controller 1105 controlsin the conventional manner a display on a display device 1107 which canbe a cathode ray tube (CRT), liquid crystal display (LCD), or any otherdisplay device. Input/output devices 1117 can include a keyboard, diskdrives, printers, a scanner, a camera, and other input and outputdevices, including a mouse or other pointing device. I/O controller 1111is coupled to one or more audio input devices 1113 such as, for example,one or more microphones.

Display controller 1105 and 110 controller 1111 can be implemented withconventional well-known technology. An audio output 1115 such as, forexample, one or more speakers, may be coupled to I/O controller 1111.Non-volatile storage 1109 is often a magnetic hard disk, an opticaldisk, or another form of storage for large amounts of data. Some of thisdata is often written, by a direct memory access process, into memory1103 during execution of software in data processing system 1100 toperform methods described herein.

One of skilled in the art will immediately recognize that the terms“computer-readable medium” and “machine-readable medium” include anytype of storage device that is accessible by processing unit 1101. Dataprocessing system 1100 can interface to external systems through a modemor network interface 1121. It will be appreciated that modem or networkinterface 1121 can be considered to be part of data processing system1100. This interface 1121 can be an analog modem, ISDN modem, cablemodem, token ring interface, satellite transmission interface, or otherinterfaces for coupling a data processing system to other dataprocessing systems.

It will be appreciated that data processing system 1100 is one exampleof many possible data processing systems which have differentarchitectures. For example, personal computers based on an Intelmicroprocessor often have multiple buses, one of which can be aninput/output (I/O) bus for the peripherals and one that directlyconnects processing unit 1101 and memory 1103 (often referred to as amemory bus). The buses are connected together through bridge componentsthat perform any necessary translation due to differing bus protocols.

Network computers are another type of data processing system that can beused with the embodiments as described herein. Network computers do notusually include a hard disk or other mass storage, and the executableprograms are loaded from a network connection into memory 1103 forexecution by processing unit 1101. A typical data processing system willusually include at least a processor, memory, and a bus coupling thememory to the processor.

It will also be appreciated that data processing system 1100 can becontrolled by operating system software which includes a file managementsystem, such as a disk operating system, which is part of the operatingsystem software. Operating system software can be the family ofoperating systems known as Macintosh® Operating System (Mac OS®) or MacOS X® from Apple Inc. of Cupertino, Calif., or the family of operatingsystems known as Windows® from Microsoft Corporation of Redmond, Wash.,and their associated file management systems. The file management systemis typically stored in non-volatile storage 1109 and causes processingunit 1101 to execute the various acts required by the operating systemto input and output data and to store data in memory, including storingfiles on non-volatile storage 1109.

In various embodiments, hardwired circuitry may be used in combinationwith software instructions to implement methods described herein. Anon-transitory machine readable medium can be used to store software anddata which when executed by a data processing system causes the systemto perform various methods described herein. This executable softwareand data may be stored in various places including for example ROM,volatile RAM, non-volatile memory, and/or cache. Portions of thissoftware and/or data may be stored in any one of these storage devices.

Thus, a machine readable medium includes any mechanism that provides(i.e., stores and/or transmits) information in a form accessible by amachine (e.g., a computer, network device, or any device with a set ofone or more processors, etc.). For example, a machine readable mediumincludes recordable/non-recordable media (e.g., read only memory (ROM);random access memory (RAM); magnetic disk storage media; optical storagemedia; flash memory devices; and the like.

The methods as described herein can be implemented using dedicatedhardware (e.g., using Field Programmable Gate Arrays, or ApplicationSpecific Integrated Circuit) or shared circuitry (e.g., microprocessorsor microcontrollers) under control of program instructions stored in amachine-readable medium. The methods as described herein can also beimplemented as computer instructions for execution on a data processingsystem, such as system 1100 of FIG. 11.

In the foregoing specification, embodiments of the invention have beendescribed with reference to specific exemplary embodiments thereof. Itwill, however, be evident that various modifications and changes may bemade thereto without departing from the broader spirit and scope of theinvention. The specification and drawings are, accordingly, to beregarded in an illustrative rather than a restrictive sense.

1-21. (canceled)
 22. An apparatus comprising: a light generating systemconfigured to produce a first set of light pulses in a first wavelengthregion and a second set of light pulses in a second wavelength region,wherein the first and the second wavelength regions do not substantiallyoverlap, and wherein the light generating system is internallyconfigured to positively chirp the first set of light pulses andnegatively chirp the second set of light pulses; a first optical systemcoupled to the light generating system, the first optical systemcomprising optics configured to combine the first set of chirped lightpulses and the second set of chirped light pulses into a set of combinedlight pulses, wherein each of the combined light pulses comprises afirst pulse from the first set of chirped light pulses and a secondpulse from the second set of chirped light pulses; and a flow cellcoupled to the first optical system and configured to convey, in a flow,particles for analysis, wherein at least one of the particles is exposedto a plurality of the combined light pulses.
 23. The apparatus of claim22, wherein the light generating system comprises temporally dispersivemeans to positively chirp the first set of light pulses and negativelychirp the second set of light pulses.
 24. The apparatus of claim 22,further comprising a synchronization system configured to providesynchronization timing signals to the light generating system, whereinthe synchronization timing signals are configured to establish a fixedphase relationship between the first set of light pulses and the secondset of light pulses.
 25. The apparatus of claim 22, wherein, for each ofthe combined light pulses, the first pulse from the first set of chirpedlight pulses is at least partially spatially and temporally overlappingwith the second pulse from the second set of chirped light pulses. 26.The apparatus of claim 22, further comprising: a second optical systemcoupled to the flow cell, the second optical system configured tocollect an interaction light signal resulting from an interaction of theplurality of the combined light pulses with the at least one of theparticles; and an analysis system comprising spectrally dispersivemeans, the analysis system configured to measure a plurality of spectralcomponents of the interaction light signal.
 26. An apparatus comprising:a light generating system configured to produce a first set of lightpulses in a first wavelength region and a second set of light pulses ina second wavelength region, wherein the first and the second wavelengthregions do not substantially overlap, and wherein the light generatingsystem is internally configured to positively chirp the first set oflight pulses and negatively chirp the second set of light pulses; afirst optical system coupled to the light generating system, the firstoptical system comprising optics configured to combine the first set ofchirped light pulses and the second set of chirped light pulses into aset of combined light pulses, wherein each of the combined light pulsescomprises a first pulse from the first set of chirped light pulses and asecond pulse from the second set of chirped light pulses; and a sampleholder coupled to the first optical system and configured to hold asample for analysis, wherein at least a portion of the sample is exposedto a plurality of the combined light pulses.
 28. The apparatus of claim27, wherein the light generating system comprises temporally dispersivemeans to positively chirp the first set of light pulses and negativelychirp the second set of light pulses.
 29. The apparatus of claim 27,further comprising a synchronization system configured to providesynchronization timing signals to the light generating system, whereinthe synchronization timing signals are configured to establish a fixedphase relationship between the first set of light pulses and the secondset of light pulses.
 30. The apparatus of claim 27, wherein, for each ofthe combined light pulses, the first pulse from the first set of chirpedlight pulses is at least partially spatially and temporally overlappingwith the second pulse from the second set of chirped light pulses. 31.The apparatus of claim 27, further comprising: a second optical systemcoupled to the sample holder, the second optical system configured tocollect an interaction light signal resulting from an interaction of theplurality of the combined light pulses with the at least a portion ofthe sample; and an analysis system comprising spectrally dispersivemeans, the analysis system configured to measure a plurality of spectralcomponents of the interaction light signal.
 32. An apparatus comprising:a light generating system configured to produce a first set of lightpulses in a first wavelength region and a second set of light pulses ina second wavelength region, wherein the first and the second wavelengthregions do not substantially overlap, and wherein the light generatingsystem is internally configured to positively chirp the first set oflight pulses and negatively chirp the second set of light pulses; afirst optical system coupled to the light generating system, the firstoptical system comprising optics configured to combine the first set ofchirped light pulses and the second set of chirped light pulses into aset of combined light pulses, wherein each of the combined light pulsescomprises a first pulse from the first set of chirped light pulses and asecond pulse from the second set of chirped light pulses; and a flowcell coupled to the first optical system and configured to convey, in aflow, particles for analysis, wherein at least one of the particles isexposed to at least one of the combined light pulses.
 33. The apparatusof claim 32, wherein the light generating system comprises temporallydispersive means to positively chirp the first set of light pulses andnegatively chirp the second set of light pulses.
 34. The apparatus ofclaim 32, further comprising: a continuous-wave light source opticallycoupled to the flow cell, the continuous-wave light source configured toconvey light to the at least one of the particles; a photodetectorconfigured to convert an optical signal resulting from an interaction oflight from the continuous-wave light source and the at least one of theparticles into an electronic detection signal; and a triggering circuitconfigured to generate a triggering signal for the light generatingsystem from the electronic detection signal.
 35. The apparatus of claim32, wherein, for each of the combined light pulses, the first pulse fromthe first set of chirped light pulses is at least partially spatiallyand temporally overlapping with the second pulse from the second set ofchirped light pulses.
 36. The apparatus of claim 32, further comprising:a second optical system coupled to the flow cell, the second opticalsystem configured to collect an interaction light signal resulting froman interaction of the at least one of the combined light pulses with theat least one of the particles; and an analysis system comprisingspectrally dispersive means, the analysis system configured to measure aplurality of spectral components of the interaction light signal.
 37. Anapparatus comprising: a light generating system configured to produce afirst set of light pulses in a first wavelength region and a second setof light pulses in a second wavelength region, wherein the first and thesecond wavelength regions do not substantially overlap, and wherein thelight generating system is internally configured to positively chirp thefirst set of light pulses and negatively chirp the second set of lightpulses; a first optical system coupled to the light generating system,the first optical system comprising optics configured to combine thefirst set of chirped light pulses and the second set of chirped lightpulses into a set of combined light pulses, wherein each of the combinedlight pulses comprises a first pulse from the first set of chirped lightpulses and a second pulse from the second set of chirped light pulses;and a sample holder coupled to the first optical system and configuredto hold a sample for analysis, wherein at least a portion of the sampleis exposed to at least one of the combined light pulses.
 38. Theapparatus of claim 37, wherein the light pulses in at least one of thefirst set and the second set of light pulses are amplified light pulses.39. The apparatus of claim 37, wherein the light generating systemcomprises temporally dispersive means to positively chirp the first setof light pulses and negatively chirp the second set of light pulses. 40.The apparatus of claim 37, wherein, for each of the combined lightpulses, the first pulse from the first set of chirped light pulses is atleast partially spatially and temporally overlapping with the secondpulse from the second set of chirped light pulses.
 41. The apparatus ofclaim 37, further comprising: a second optical system coupled to thesample holder, the second optical system configured to collect aninteraction light signal resulting from an interaction of the at leastone of the combined light pulses with the at least a portion of thesample; and an analysis system comprising spectrally dispersive means,the analysis system configured to measure a plurality of spectralcomponents of the interaction light signal.